A Genome-Scale Investigation of How Sequence, Function, and Tree-Based Gene Properties Influence Phylogenetic Inference.

PubMed

Shen, Xing-Xing; Salichos, Leonidas; Rokas, Antonis

2016-09-02

Molecular phylogenetic inference is inherently dependent on choices in both methodology and data. Many insightful studies have shown how choices in methodology, such as the model of sequence evolution or optimality criterion used, can strongly influence inference. In contrast, much less is known about the impact of choices in the properties of the data, typically genes, on phylogenetic inference. We investigated the relationships between 52 gene properties (24 sequence-based, 19 function-based, and 9 tree-based) with each other and with three measures of phylogenetic signal in two assembled data sets of 2,832 yeast and 2,002 mammalian genes. We found that most gene properties, such as evolutionary rate (measured through the percent average of pairwise identity across taxa) and total tree length, were highly correlated with each other. Similarly, several gene properties, such as gene alignment length, Guanine-Cytosine content, and the proportion of tree distance on internal branches divided by relative composition variability (treeness/RCV), were strongly correlated with phylogenetic signal. Analysis of partial correlations between gene properties and phylogenetic signal in which gene evolutionary rate and alignment length were simultaneously controlled, showed similar patterns of correlations, albeit weaker in strength. Examination of the relative importance of each gene property on phylogenetic signal identified gene alignment length, alongside with number of parsimony-informative sites and variable sites, as the most important predictors. Interestingly, the subsets of gene properties that optimally predicted phylogenetic signal differed considerably across our three phylogenetic measures and two data sets; however, gene alignment length and RCV were consistently included as predictors of all three phylogenetic measures in both yeasts and mammals. These results suggest that a handful of sequence-based gene properties are reliable predictors of phylogenetic signal and could be useful in guiding the choice of phylogenetic markers. © The Author 2016. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.

Molecular tandem repeat strategy for elucidating mechanical properties of high-strength proteins

PubMed Central

Jung, Huihun; Pena-Francesch, Abdon; Saadat, Alham; Sebastian, Aswathy; Kim, Dong Hwan; Hamilton, Reginald F.; Albert, Istvan; Allen, Benjamin D.; Demirel, Melik C.

2016-01-01

Many globular and structural proteins have repetitions in their sequences or structures. However, a clear relationship between these repeats and their contribution to the mechanical properties remains elusive. We propose a new approach for the design and production of synthetic polypeptides that comprise one or more tandem copies of a single unit with distinct amorphous and ordered regions. Our designed sequences are based on a structural protein produced in squid suction cups that has a segmented copolymer structure with amorphous and crystalline domains. We produced segmented polypeptides with varying repeat number, while keeping the lengths and compositions of the amorphous and crystalline regions fixed. We showed that mechanical properties of these synthetic proteins could be tuned by modulating their molecular weights. Specifically, the toughness and extensibility of synthetic polypeptides increase as a function of the number of tandem repeats. This result suggests that the repetitions in native squid proteins could have a genetic advantage for increased toughness and flexibility. PMID:27222581

Complete Genome Sequence of Bifidobacterium bifidum S17▿

PubMed Central

Zhurina, Daria; Zomer, Aldert; Gleinser, Marita; Brancaccio, Vincenco Francesco; Auchter, Marc; Waidmann, Mark S.; Westermann, Christina; van Sinderen, Douwe; Riedel, Christian U.

2011-01-01

Here, we report on the first completely annotated genome sequence of a Bifidobacterium bifidum strain. B. bifidum S17, isolated from feces of a breast-fed infant, was shown to strongly adhere to intestinal epithelial cells and has potent anti-inflammatory activity in vitro and in vivo. The genome sequence will provide new insights into the biology of this potential probiotic organism and allow for the characterization of the molecular mechanisms underlying its beneficial properties. PMID:21037011

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

PubMed

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

2016-07-01

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

Four new topological indices based on the molecular path code.

PubMed

Balaban, Alexandru T; Beteringhe, Adrian; Constantinescu, Titus; Filip, Petru A; Ivanciuc, Ovidiu

2007-01-01

The sequence of all paths pi of lengths i = 1 to the maximum possible length in a hydrogen-depleted molecular graph (which sequence is also called the molecular path code) contains significant information on the molecular topology, and as such it is a reasonable choice to be selected as the basis of topological indices (TIs). Four new (or five partly new) TIs with progressively improved performance (judged by correctly reflecting branching, centricity, and cyclicity of graphs, ordering of alkanes, and low degeneracy) have been explored. (i) By summing the squares of all numbers in the sequence one obtains Sigmaipi(2), and by dividing this sum by one plus the cyclomatic number, a Quadratic TI is obtained: Q = Sigmaipi(2)/(mu+1). (ii) On summing the Square roots of all numbers in the sequence one obtains Sigmaipi(1/2), and by dividing this sum by one plus the cyclomatic number, the TI denoted by S is obtained: S = Sigmaipi(1/2)/(mu+1). (iii) On dividing terms in this sum by the corresponding topological distances, one obtains the Distance-reduced index D = Sigmai{pi(1/2)/[i(mu+1)]}. Two similar formulas define the next two indices, the first one with no square roots: (iv) distance-Attenuated index: A = Sigmai{pi/[i(mu + 1)]}; and (v) the last TI with two square roots: Path-count index: P = Sigmai{pi(1/2)/[i(1/2)(mu + 1)]}. These five TIs are compared for their degeneracy, ordering of alkanes, and performance in QSPR (for all alkanes with 3-12 carbon atoms and for all possible chemical cyclic or acyclic graphs with 4-6 carbon atoms) in correlations with six physical properties and one chemical property.

Linear Relation for Wind-blown Bubble Sizes of Main-sequence OB Stars in a Molecular Environment and Implication for Supernova Progenitors

NASA Astrophysics Data System (ADS)

Chen, Yang; Zhou, Ping; Chu, You-Hua

2013-05-01

We find a linear relationship between the size of a massive star's main-sequence bubble in a molecular environment and the star's initial mass: R b ≈ 1.22 M/M ⊙ - 9.16 pc, assuming a constant interclump pressure. Since stars in the mass range of 8 to 25-30 M ⊙ will end their evolution in the red supergiant phase without launching a Wolf-Rayet wind, the main-sequence wind-blown bubbles are mainly responsible for the extent of molecular gas cavities, while the effect of the photoionization is comparatively small. This linear relation can thus be used to infer the masses of the massive star progenitors of supernova remnants (SNRs) that are discovered to evolve in molecular cavities, while few other means are available for inferring the properties of SNR progenitors. We have used this method to estimate the initial masses of the progenitors of eight SNRs: Kes 69, Kes 75, Kes 78, 3C 396, 3C 397, HC 40, Vela, and RX J1713-3946.

Engineering of a target site-specific recombinase by a combined evolution- and structure-guided approach

PubMed Central

Abi-Ghanem, Josephine; Chusainow, Janet; Karimova, Madina; Spiegel, Christopher; Hofmann-Sieber, Helga; Hauber, Joachim; Buchholz, Frank; Pisabarro, M. Teresa

2013-01-01

Site-specific recombinases (SSRs) can perform DNA rearrangements, including deletions, inversions and translocations when their naive target sequences are placed strategically into the genome of an organism. Hence, in order to employ SSRs in heterologous hosts, their target sites have to be introduced into the genome of an organism before the enzyme can be practically employed. Engineered SSRs hold great promise for biotechnology and advanced biomedical applications, as they promise to extend the usefulness of SSRs to allow efficient and specific recombination of pre-existing, natural genomic sequences. However, the generation of enzymes with desired properties remains challenging. Here, we use substrate-linked directed evolution in combination with molecular modeling to rationally engineer an efficient and specific recombinase (sTre) that readily and specifically recombines a sequence present in the HIV-1 genome. We elucidate the role of key residues implicated in the molecular recognition mechanism and we present a rationale for sTre’s enhanced specificity. Combining evolutionary and rational approaches should help in accelerating the generation of enzymes with desired properties for use in biotechnology and biomedicine. PMID:23275541

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

PubMed

Kumari, Archana; Akkoç, Nefise; Akçelik, Mustafa

2012-04-01

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

Sequence analysis of serum albumins reveals the molecular evolution of ligand recognition properties.

PubMed

Fanali, Gabriella; Ascenzi, Paolo; Bernardi, Giorgio; Fasano, Mauro

2012-01-01

Serum albumin (SA) is a circulating protein providing a depot and carrier for many endogenous and exogenous compounds. At least seven major binding sites have been identified by structural and functional investigations mainly in human SA. SA is conserved in vertebrates, with at least 49 entries in protein sequence databases. The multiple sequence analysis of this set of entries leads to the definition of a cladistic tree for the molecular evolution of SA orthologs in vertebrates, thus showing the clustering of the considered species, with lamprey SAs (Lethenteron japonicum and Petromyzon marinus) in a separate outgroup. Sequence analysis aimed at searching conserved domains revealed that most SA sequences are made up by three repeated domains (about 600 residues), as extensively characterized for human SA. On the contrary, lamprey SAs are giant proteins (about 1400 residues) comprising seven repeated domains. The phylogenetic analysis of the SA family reveals a stringent correlation with the taxonomic classification of the species available in sequence databases. A focused inspection of the sequences of ligand binding sites in SA revealed that in all sites most residues involved in ligand binding are conserved, although the versatility towards different ligands could be peculiar of higher organisms. Moreover, the analysis of molecular links between the different sites suggests that allosteric modulation mechanisms could be restricted to higher vertebrates.

Course 6: Star Formation

NASA Astrophysics Data System (ADS)

Natta, A.

Contents 1 Introduction 2 Collapse of molecular cores 2.1 Giant molecular clouds and cores 2.2 Conditions for collapse 2.3 Free-fall collapse 2.4 Collapse of an isothermal sphere of gas 2.5 Collapse of a slowly rotating core 3 Observable properties of protostars 3.1 Evidence of infall from molecular line profiles 3.2 SEDs of protostars 3.3 The line spectrumof a protostar 4 Protostellar and pre-main-sequence evolution 4.1 The protostellar phase 4.2 Pre-main-sequence evolution 4.3 The birthline 5 Circumstellar disks 5.1 Accretion disks 5.2 Properties of steady accretion disks 5.3 Reprocessing disks 5.4 Disk-star interaction 6 SEDs of disks 6.1 Power-law disks 6.2 Long-wavelength flux and disk mass 6.3 Comparison with TTS observations: Heating mechanism 7 Disk properties from observations 7.1 Mass accretion rate 7.2 Inner radius 7.3 Masses 7.4 Sizes 8 Disk lifetimes 8.1 Ground-based near and mid-infrared surveys 8.2 Mid-infrared ISOCAMsurveys 8.3 ISOPHOT 60 microm survey 8.4 Surveys at millimeter wavelengths 9 Disk evolution 9.1 Can we observe the early planet formation phase? 9.2 Evidence for grain growth 9.3 Evidence of planetesimals 9.4 Where is the diskmass? 10 Secondary or debris disks 11 Summary

Four-step reaction for polytriazine elastomers

NASA Technical Reports Server (NTRS)

Rosser, R. W.; Korus, R. A.

1980-01-01

Four step imidoylamidine reaction sequence is used to make crosslinked polyperfluoralkyltriazines with superior elastomeric properties, greater molecular weight, and crosslinking control. Polymers can find useful application in fuel tank sealants, o-ring, wire enamels, pneumatic ducts, and many other applications.

Synthetic Molecular Evolution of Membrane-Active Peptides

NASA Astrophysics Data System (ADS)

Wimley, William

The physical chemistry of membrane partitioning largely determines the function of membrane active peptides. Membrane-active peptides have potential utility in many areas, including in the cellular delivery of polar compounds, cancer therapy, biosensor design, and in antibacterial, antiviral and antifungal therapies. Yet, despite decades of research on thousands of known examples, useful sequence-structure-function relationships are essentially unknown. Because peptide-membrane interactions within the highly fluid bilayer are dynamic and heterogeneous, accounts of mechanism are necessarily vague and descriptive, and have little predictive power. This creates a significant roadblock to advances in the field. We are bypassing that roadblock with synthetic molecular evolution: iterative peptide library design and orthogonal high-throughput screening. We start with template sequences that have at least some useful activity, and create small, focused libraries using structural and biophysical principles to design the sequence space around the template. Orthogonal high-throughput screening is used to identify gain-of-function peptides by simultaneously selecting for several different properties (e.g. solubility, activity and toxicity). Multiple generations of iterative library design and screening have enabled the identification of membrane-active sequences with heretofore unknown properties, including clinically relevant, broad-spectrum activity against drug-resistant bacteria and enveloped viruses as well as pH-triggered macromolecular poration.

Molecular studies in olive (Olea europaea L.): overview on DNA markers applications and recent advances in genome analysis.

PubMed

Bracci, T; Busconi, M; Fogher, C; Sebastiani, L

2011-04-01

Olive (Olea europaea L.) is one of the oldest agricultural tree crops worldwide and is an important source of oil with beneficial properties for human health. This emblematic tree crop of the Mediterranean Basin, which has conserved a very wide germplasm estimated in more than 1,200 cultivars, is a diploid species (2n = 2x = 46) that is present in two forms, namely wild (Olea europaea subsp. europaea var. sylvestris) and cultivated (Olea europaea subsp. europaea var. europaea). In spite of its economic and nutritional importance, there are few data about the genetic of olive if compared with other fruit crops. Available molecular data are especially related to the application of molecular markers to the analysis of genetic variability in Olea europaea complex and to develop efficient molecular tools for the olive oil origin traceability. With regard to genomic research, in the last years efforts are made for the identification of expressed sequence tag, with particular interest in those sequences expressed during fruit development and in pollen allergens. Very recently the sequencing of chloroplast genome provided new information on the olive nucleotide sequence, opening the olive genomic era. In this article, we provide an overview of the most relevant results in olive molecular studies. A particular attention was given to DNA markers and their application that constitute the most part of published researches. The first important results in genome analysis were reported.

Modular protein domains: an engineering approach toward functional biomaterials.

PubMed

Lin, Charng-Yu; Liu, Julie C

2016-08-01

Protein domains and peptide sequences are a powerful tool for conferring specific functions to engineered biomaterials. Protein sequences with a wide variety of functionalities, including structure, bioactivity, protein-protein interactions, and stimuli responsiveness, have been identified, and advances in molecular biology continue to pinpoint new sequences. Protein domains can be combined to make recombinant proteins with multiple functionalities. The high fidelity of the protein translation machinery results in exquisite control over the sequence of recombinant proteins and the resulting properties of protein-based materials. In this review, we discuss protein domains and peptide sequences in the context of functional protein-based materials, composite materials, and their biological applications. Copyright © 2016 Elsevier Ltd. All rights reserved.

Positive selection in octopus haemocyanin indicates functional links to temperature adaptation.

PubMed

Oellermann, Michael; Strugnell, Jan M; Lieb, Bernhard; Mark, Felix C

2015-07-05

Octopods have successfully colonised the world's oceans from the tropics to the poles. Yet, successful persistence in these habitats has required adaptations of their advanced physiological apparatus to compensate impaired oxygen supply. Their oxygen transporter haemocyanin plays a major role in cold tolerance and accordingly has undergone functional modifications to sustain oxygen release at sub-zero temperatures. However, it remains unknown how molecular properties evolved to explain the observed functional adaptations. We thus aimed to assess whether natural selection affected molecular and structural properties of haemocyanin that explains temperature adaptation in octopods. Analysis of 239 partial sequences of the haemocyanin functional units (FU) f and g of 28 octopod species of polar, temperate, subtropical and tropical origin revealed natural selection was acting primarily on charge properties of surface residues. Polar octopods contained haemocyanins with higher net surface charge due to decreased glutamic acid content and higher numbers of basic amino acids. Within the analysed partial sequences, positive selection was present at site 2545, positioned between the active copper binding centre and the FU g surface. At this site, methionine was the dominant amino acid in polar octopods and leucine was dominant in tropical octopods. Sites directly involved in oxygen binding or quaternary interactions were highly conserved within the analysed sequence. This study has provided the first insight into molecular and structural mechanisms that have enabled octopods to sustain oxygen supply from polar to tropical conditions. Our findings imply modulation of oxygen binding via charge-charge interaction at the protein surface, which stabilize quaternary interactions among functional units to reduce detrimental effects of high pH on venous oxygen release. Of the observed partial haemocyanin sequence, residue 2545 formed a close link between the FU g surface and the active centre, suggesting a role as allosteric binding site. The prevalence of methionine at this site in polar octopods, implies regulation of oxygen affinity via increased sensitivity to allosteric metal binding. High sequence conservation of sites directly involved in oxygen binding indicates that functional modifications of octopod haemocyanin rather occur via more subtle mechanisms, as observed in this study.

Next generation sequencing and de novo transcriptome analysis of Costus pictus D. Don, a non-model plant with potent anti-diabetic properties

PubMed Central

2012-01-01

Background Phyto-remedies for diabetic control are popular among patients with Type II Diabetes mellitus (DM), in addition to other diabetic control measures. A number of plant species are known to possess diabetic control properties. Costus pictus D. Don is popularly known as “Insulin Plant” in Southern India whose leaves have been reported to increase insulin pools in blood plasma. Next Generation Sequencing is employed as a powerful tool for identifying molecular signatures in the transcriptome related to physiological functions of plant tissues. We sequenced the leaf transcriptome of C. pictus using Illumina reversible dye terminator sequencing technology and used combination of bioinformatics tools for identifying transcripts related to anti-diabetic properties of C. pictus. Results A total of 55,006 transcripts were identified, of which 69.15% transcripts could be annotated. We identified transcripts related to pathways of bixin biosynthesis and geraniol and geranial biosynthesis as major transcripts from the class of isoprenoid secondary metabolites and validated the presence of putative norbixin methyltransferase, a precursor of Bixin. The transcripts encoding these terpenoids are known to be Peroxisome Proliferator-Activated Receptor (PPAR) agonists and anti-glycation agents. Sequential extraction and High Performance Liquid Chromatography (HPLC) confirmed the presence of bixin in C. pictus methanolic extracts. Another significant transcript identified in relation to anti-diabetic, anti-obesity and immuno-modulation is of Abscisic Acid biosynthetic pathway. We also report many other transcripts for the biosynthesis of antitumor, anti-oxidant and antimicrobial metabolites of C. pictus leaves. Conclusion Solid molecular signatures (transcripts related to bixin, abscisic acid, and geranial and geraniol biosynthesis) for the anti-diabetic properties of C. pictus leaves and vital clues related to the other phytochemical functions like antitumor, anti-oxidant, immuno-modulatory, anti-microbial and anti-malarial properties through the secondary metabolite pathway annotations are reported. The data provided will be of immense help to researchers working in the treatment of DM using herbal therapies. PMID:23176672

Occurrence and molecular characterization of Cryptosporidium spp. isolated from domestic animals in a rural area surrounding Atlantic dry forest fragments in Teodoro Sampaio municipality, State of São Paulo, Brazil.

PubMed

Sevá, Anaiá da Paixão; Funada, Mikaela Renata; Souza, Sheila de Oliveira; Nava, Alessandra; Richtzenhain, Leonardo José; Soares, Rodrigo Martins

2010-01-01

The aim of this study was to assess the occurrence of Cryptosporidium in domestic animals in rural properties surrounding rain forest fragments within the municipality of Teodoro Sampaio, southeastern Brazil. Conventional sucrose flotation method followed by molecular characterization of the parasites by sequencing PCR products amplified from SSU rRNA gene were used. Stool samples were collected from domestic animals raised as pets and livestock in all rural properties surrounding three forest fragments. Samples from cattle (197), equine (63), pigs (25), sheep (11), and dogs (28) were collected from 98 rural properties. The frequency of occurrence of Cryptosporidium within each animal species was 3.0% (6/197) among cattle and 10.7% (3/28) among dogs. Cryptosporidium was not detected in stool samples from equine, sheep, and pigs. All sequences obtained from the six samples of calves showed molecular identity with Cryptosporidium andersoni while all sequences from dog samples were similar to C. canis. The frequency of occurrence of Cryptosporidium in these domestic animal species was low. The absence of C. parvum in the present study suggests that the zoonotic cycle of cryptosporidiosis may not be relevant in the region studied. The presence of Cryptosporidium species seldom described in humans may be, otherwise, important for the wild fauna as these animals are a source of infection and dissemination of this protozoan to other animal species. The impact and magnitude of infection by C. andersoni in wild ruminants and C. canis in wild canids have to be assessed in future studies to better understand the actual importance of these species in this region.

Two-dimensional honeycomb network through sequence-controlled self-assembly of oligopeptides.

PubMed

Abb, Sabine; Harnau, Ludger; Gutzler, Rico; Rauschenbach, Stephan; Kern, Klaus

2016-01-12

The sequence of a peptide programs its self-assembly and hence the expression of specific properties through non-covalent interactions. A large variety of peptide nanostructures has been designed employing different aspects of these non-covalent interactions, such as dispersive interactions, hydrogen bonding or ionic interactions. Here we demonstrate the sequence-controlled fabrication of molecular nanostructures using peptides as bio-organic building blocks for two-dimensional (2D) self-assembly. Scanning tunnelling microscopy reveals changes from compact or linear assemblies (angiotensin I) to long-range ordered, chiral honeycomb networks (angiotensin II) as a result of removal of steric hindrance by sequence modification. Guided by our observations, molecular dynamic simulations yield atomistic models for the elucidation of interpeptide-binding motifs. This new approach to 2D self-assembly on surfaces grants insight at the atomic level that will enable the use of oligo- and polypeptides as large, multi-functional bio-organic building blocks, and opens a new route towards rationally designed, bio-inspired surfaces.

'Candidatus Phytoplasma solani', a novel taxon associated with stolbur- and bois noir-related diseases of plants.

PubMed

Quaglino, Fabio; Zhao, Yan; Casati, Paola; Bulgari, Daniela; Bianco, Piero Attilio; Wei, Wei; Davis, Robert Edward

2013-08-01

Phytoplasmas classified in group 16SrXII infect a wide range of plants and are transmitted by polyphagous planthoppers of the family Cixiidae. Based on 16S rRNA gene sequence identity and biological properties, group 16SrXII encompasses several species, including 'Candidatus Phytoplasma australiense', 'Candidatus Phytoplasma japonicum' and 'Candidatus Phytoplasma fragariae'. Other group 16SrXII phytoplasma strains are associated with stolbur disease in wild and cultivated herbaceous and woody plants and with bois noir disease in grapevines (Vitis vinifera L.). Such latter strains have been informally proposed to represent a separate species, 'Candidatus Phytoplasma solani', but a formal description of this taxon has not previously been published. In the present work, stolbur disease strain STOL11 (STOL) was distinguished from reference strains of previously described species of the 'Candidatus Phytoplasma' genus based on 16S rRNA gene sequence similarity and a unique signature sequence in the 16S rRNA gene. Other stolbur- and bois noir-associated ('Ca. Phytoplasma solani') strains shared >99 % 16S rRNA gene sequence similarity with strain STOL11 and contained the signature sequence. 'Ca. Phytoplasma solani' is the only phytoplasma known to be transmitted by Hyalesthes obsoletus. Insect vectorship and molecular characteristics are consistent with the concept that diverse 'Ca. Phytoplasma solani' strains share common properties and represent an ecologically distinct gene pool. Phylogenetic analyses of 16S rRNA, tuf, secY and rplV-rpsC gene sequences supported this view and yielded congruent trees in which 'Ca. Phytoplasma solani' strains formed, within the group 16SrXII clade, a monophyletic subclade that was most closely related to, but distinct from, that of 'Ca. Phytoplasma australiense'-related strains. Based on distinct molecular and biological properties, stolbur- and bois noir-associated strains are proposed to represent a novel species level taxon, 'Ca. Phytoplasma solani'; STOL11 is designated the reference strain.

Topological Structure of the Space of Phenotypes: The Case of RNA Neutral Networks

PubMed Central

Aguirre, Jacobo; Buldú, Javier M.; Stich, Michael; Manrubia, Susanna C.

2011-01-01

The evolution and adaptation of molecular populations is constrained by the diversity accessible through mutational processes. RNA is a paradigmatic example of biopolymer where genotype (sequence) and phenotype (approximated by the secondary structure fold) are identified in a single molecule. The extreme redundancy of the genotype-phenotype map leads to large ensembles of RNA sequences that fold into the same secondary structure and can be connected through single-point mutations. These ensembles define neutral networks of phenotypes in sequence space. Here we analyze the topological properties of neutral networks formed by 12-nucleotides RNA sequences, obtained through the exhaustive folding of sequence space. A total of 412 sequences fragments into 645 subnetworks that correspond to 57 different secondary structures. The topological analysis reveals that each subnetwork is far from being random: it has a degree distribution with a well-defined average and a small dispersion, a high clustering coefficient, and an average shortest path between nodes close to its minimum possible value, i.e. the Hamming distance between sequences. RNA neutral networks are assortative due to the correlation in the composition of neighboring sequences, a feature that together with the symmetries inherent to the folding process explains the existence of communities. Several topological relationships can be analytically derived attending to structural restrictions and generic properties of the folding process. The average degree of these phenotypic networks grows logarithmically with their size, such that abundant phenotypes have the additional advantage of being more robust to mutations. This property prevents fragmentation of neutral networks and thus enhances the navigability of sequence space. In summary, RNA neutral networks show unique topological properties, unknown to other networks previously described. PMID:22028856

Tracking the Molecular Evolution of Calcium Permeability in a Nicotinic Acetylcholine Receptor

PubMed Central

Lipovsek, Marcela; Fierro, Angélica; Pérez, Edwin G.; Boffi, Juan C.; Millar, Neil S.; Fuchs, Paul A.; Katz, Eleonora; Elgoyhen, Ana Belén

2014-01-01

Nicotinic acetylcholine receptors are a family of ligand-gated nonselective cationic channels that participate in fundamental physiological processes at both the central and the peripheral nervous system. The extent of calcium entry through ligand-gated ion channels defines their distinct functions. The α9α10 nicotinic cholinergic receptor, expressed in cochlear hair cells, is a peculiar member of the family as it shows differences in the extent of calcium permeability across species. In particular, mammalian α9α10 receptors are among the ligand-gated ion channels which exhibit the highest calcium selectivity. This acquired differential property provides the unique opportunity of studying how protein function was shaped along evolutionary history, by tracking its evolutionary record and experimentally defining the amino acid changes involved. We have applied a molecular evolution approach of ancestral sequence reconstruction, together with molecular dynamics simulations and an evolutionary-based mutagenesis strategy, in order to trace the molecular events that yielded a high calcium permeable nicotinic α9α10 mammalian receptor. Only three specific amino acid substitutions in the α9 subunit were directly involved. These are located at the extracellular vestibule and at the exit of the channel pore and not at the transmembrane region 2 of the protein as previously thought. Moreover, we show that these three critical substitutions only increase calcium permeability in the context of the mammalian but not the avian receptor, stressing the relevance of overall protein structure on defining functional properties. These results highlight the importance of tracking evolutionarily acquired changes in protein sequence underlying fundamental functional properties of ligand-gated ion channels. PMID:25193338

De Novo Transcriptome Sequencing Reveals Important Molecular Networks and Metabolic Pathways of the Plant, Chlorophytum borivilianum

PubMed Central

Kalra, Shikha; Puniya, Bhanwar Lal; Kulshreshtha, Deepika; Kumar, Sunil; Kaur, Jagdeep; Ramachandran, Srinivasan; Singh, Kashmir

2013-01-01

Chlorophytum borivilianum, an endangered medicinal plant species is highly recognized for its aphrodisiac properties provided by saponins present in the plant. The transcriptome information of this species is limited and only few hundred expressed sequence tags (ESTs) are available in the public databases. To gain molecular insight of this plant, high throughput transcriptome sequencing of leaf RNA was carried out using Illumina's HiSeq 2000 sequencing platform. A total of 22,161,444 single end reads were retrieved after quality filtering. Available (e.g., De-Bruijn/Eulerian graph) and in-house developed bioinformatics tools were used for assembly and annotation of transcriptome. A total of 101,141 assembled transcripts were obtained, with coverage size of 22.42 Mb and average length of 221 bp. Guanine-cytosine (GC) content was found to be 44%. Bioinformatics analysis, using non-redundant proteins, gene ontology (GO), enzyme commission (EC) and kyoto encyclopedia of genes and genomes (KEGG) databases, extracted all the known enzymes involved in saponin and flavonoid biosynthesis. Few genes of the alkaloid biosynthesis, along with anticancer and plant defense genes, were also discovered. Additionally, several cytochrome P450 (CYP450) and glycosyltransferase unique sequences were also found. We identified simple sequence repeat motifs in transcripts with an abundance of di-nucleotide simple sequence repeat (SSR; 43.1%) markers. Large scale expression profiling through Reads per Kilobase per Million mapped reads (RPKM) showed major genes involved in different metabolic pathways of the plant. Genes, expressed sequence tags (ESTs) and unique sequences from this study provide an important resource for the scientific community, interested in the molecular genetics and functional genomics of C. borivilianum. PMID:24376689

De Novo transcriptome sequencing reveals important molecular networks and metabolic pathways of the plant, Chlorophytum borivilianum.

PubMed

Kalra, Shikha; Puniya, Bhanwar Lal; Kulshreshtha, Deepika; Kumar, Sunil; Kaur, Jagdeep; Ramachandran, Srinivasan; Singh, Kashmir

2013-01-01

Chlorophytum borivilianum, an endangered medicinal plant species is highly recognized for its aphrodisiac properties provided by saponins present in the plant. The transcriptome information of this species is limited and only few hundred expressed sequence tags (ESTs) are available in the public databases. To gain molecular insight of this plant, high throughput transcriptome sequencing of leaf RNA was carried out using Illumina's HiSeq 2000 sequencing platform. A total of 22,161,444 single end reads were retrieved after quality filtering. Available (e.g., De-Bruijn/Eulerian graph) and in-house developed bioinformatics tools were used for assembly and annotation of transcriptome. A total of 101,141 assembled transcripts were obtained, with coverage size of 22.42 Mb and average length of 221 bp. Guanine-cytosine (GC) content was found to be 44%. Bioinformatics analysis, using non-redundant proteins, gene ontology (GO), enzyme commission (EC) and kyoto encyclopedia of genes and genomes (KEGG) databases, extracted all the known enzymes involved in saponin and flavonoid biosynthesis. Few genes of the alkaloid biosynthesis, along with anticancer and plant defense genes, were also discovered. Additionally, several cytochrome P450 (CYP450) and glycosyltransferase unique sequences were also found. We identified simple sequence repeat motifs in transcripts with an abundance of di-nucleotide simple sequence repeat (SSR; 43.1%) markers. Large scale expression profiling through Reads per Kilobase per Million mapped reads (RPKM) showed major genes involved in different metabolic pathways of the plant. Genes, expressed sequence tags (ESTs) and unique sequences from this study provide an important resource for the scientific community, interested in the molecular genetics and functional genomics of C. borivilianum.

Complementary molecular information changes our perception of food web structure

PubMed Central

Wirta, Helena K.; Hebert, Paul D. N.; Kaartinen, Riikka; Prosser, Sean W.; Várkonyi, Gergely; Roslin, Tomas

2014-01-01

How networks of ecological interactions are structured has a major impact on their functioning. However, accurately resolving both the nodes of the webs and the links between them is fraught with difficulties. We ask whether the new resolution conferred by molecular information changes perceptions of network structure. To probe a network of antagonistic interactions in the High Arctic, we use two complementary sources of molecular data: parasitoid DNA sequenced from the tissues of their hosts and host DNA sequenced from the gut of adult parasitoids. The information added by molecular analysis radically changes the properties of interaction structure. Overall, three times as many interaction types were revealed by combining molecular information from parasitoids and hosts with rearing data, versus rearing data alone. At the species level, our results alter the perceived host specificity of parasitoids, the parasitoid load of host species, and the web-wide role of predators with a cryptic lifestyle. As the northernmost network of host–parasitoid interactions quantified, our data point exerts high leverage on global comparisons of food web structure. However, how we view its structure will depend on what information we use: compared with variation among networks quantified at other sites, the properties of our web vary as much or much more depending on the techniques used to reconstruct it. We thus urge ecologists to combine multiple pieces of evidence in assessing the structure of interaction webs, and suggest that current perceptions of interaction structure may be strongly affected by the methods used to construct them. PMID:24449902

Controlling charge current through a DNA based molecular transistor

NASA Astrophysics Data System (ADS)

Behnia, S.; Fathizadeh, S.; Ziaei, J.

2017-01-01

Molecular electronics is complementary to silicon-based electronics and may induce electronic functions which are difficult to obtain with conventional technology. We have considered a DNA based molecular transistor and study its transport properties. The appropriate DNA sequence as a central chain in molecular transistor and the functional interval for applied voltages is obtained. I-V characteristic diagram shows the rectifier behavior as well as the negative differential resistance phenomenon of DNA transistor. We have observed the nearly periodic behavior in the current flowing through DNA. It is reported that there is a critical gate voltage for each applied bias which above it, the electrical current is always positive.

NABIC marker database: A molecular markers information network of agricultural crops.

PubMed

Kim, Chang-Kug; Seol, Young-Joo; Lee, Dong-Jun; Jeong, In-Seon; Yoon, Ung-Han; Lee, Gang-Seob; Hahn, Jang-Ho; Park, Dong-Suk

2013-01-01

In 2013, National Agricultural Biotechnology Information Center (NABIC) reconstructs a molecular marker database for useful genetic resources. The web-based marker database consists of three major functional categories: map viewer, RSN marker and gene annotation. It provides 7250 marker locations, 3301 RSN marker property, 3280 molecular marker annotation information in agricultural plants. The individual molecular marker provides information such as marker name, expressed sequence tag number, gene definition and general marker information. This updated marker-based database provides useful information through a user-friendly web interface that assisted in tracing any new structures of the chromosomes and gene positional functions using specific molecular markers. The database is available for free at http://nabic.rda.go.kr/gere/rice/molecularMarkers/

Purification and properties of a third form of anthranilate-5-phosphoribosylpyrophosphate phosphoribosyltransferase from the enterobacteriaceae

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

Largen, M.; Mills, S.E.; Rowe, J.

1978-01-25

Anthranilate-5-phosphoribosypyrophosphate phosphoribosyltransferase was purified from the bacterium Erwinia carotovora, a member of the Enterobacteriaceae. The enzyme was homogeneous according to the criteria of gel electrophoresis and NH/sub 2/-terminal amino acid sequence analysis. The molecular weight of the enzyme as determined on a calibrated Sephadex G-200 column was 67,000 +- 2,000. Sodium dodecyl sulfate-polyacrylamide gels gave a subunit molecular weight of 40,000 +- 1,000, suggesting that the enzyme was a dimer. A comparison of the NH/sub 2/-terminal sequence of the enzyme with the (previously determined) homologue from Serratia marcescens, a monomer with a molecular weight of 45,000, showed that the largermore » Serratia subunit came into register with amino acid 14 of the Erwinia subunit. The register for the length of the known overlap, 26 amino acids, was highly conserved.« less

Binding of anti-apoptotic Bcl-2 with different BH3 peptides: A molecular dynamics study

NASA Astrophysics Data System (ADS)

Zhang, Dawei; Liu, Huihui; Cui, Jinglan

2018-01-01

In this work, molecular dynamics simulation and free energy calculations are utilized to study how different BH3 peptides originating from Bax, Bim, Bik and Noxa interact with Bcl-2, one of the main members of anti-apoptotic proteins. The effects of peptide length, sequence and helical content on the binding affinity are discussed, on which a novel BH3-like peptide is designed in silico with an improved binding property.

The Coding of Biological Information: From Nucleotide Sequence to Protein Recognition

NASA Astrophysics Data System (ADS)

Štambuk, Nikola

The paper reviews the classic results of Swanson, Dayhoff, Grantham, Blalock and Root-Bernstein, which link genetic code nucleotide patterns to the protein structure, evolution and molecular recognition. Symbolic representation of the binary addresses defining particular nucleotide and amino acid properties is discussed, with consideration of: structure and metric of the code, direct correspondence between amino acid and nucleotide information, and molecular recognition of the interacting protein motifs coded by the complementary DNA and RNA strands.

Improving protein complex classification accuracy using amino acid composition profile.

PubMed

Huang, Chien-Hung; Chou, Szu-Yu; Ng, Ka-Lok

2013-09-01

Protein complex prediction approaches are based on the assumptions that complexes have dense protein-protein interactions and high functional similarity between their subunits. We investigated those assumptions by studying the subunits' interaction topology, sequence similarity and molecular function for human and yeast protein complexes. Inclusion of amino acids' physicochemical properties can provide better understanding of protein complex properties. Principal component analysis is carried out to determine the major features. Adopting amino acid composition profile information with the SVM classifier serves as an effective post-processing step for complexes classification. Improvement is based on primary sequence information only, which is easy to obtain. Copyright © 2013 Elsevier Ltd. All rights reserved.

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

PubMed

Song, Jiangning; Burrage, Kevin

2006-10-03

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

Identification of Single-Copy Orthologous Genes between Physalis and Solanum lycopersicum and Analysis of Genetic Diversity in Physalis Using Molecular Markers

PubMed Central

Wei, Jingli; Hu, Xiaorong; Yang, Jingjing; Yang, Wencai

2012-01-01

The genus Physalis includes a number of commercially important edible and ornamental species. Its high nutritional value and potential medicinal properties leads to the increased commercial interest in the products of this genus worldwide. However, lack of molecular markers prevents the detailed study of genetics and phylogeny in Physalis, which limits the progress of breeding. In the present study, we compared the DNA sequences between Physalis and tomato, and attempted to analyze genetic diversity in Physalis using tomato markers. Blasting 23180 DNA sequences derived from Physalis against the International Tomato Annotation Group (ITAG) Release2.3 Predicted CDS (SL2.40) discovered 3356 single-copy orthologous genes between them. A total of 38 accessions from at least six species of Physalis were subjected to genetic diversity analysis using 97 tomato markers and 25 SSR markers derived from P. peruviana. Majority (73.2%) of tomato markers could amplify DNA fragments from at least one accession of Physalis. Diversity in Physalis at molecular level was also detected. The average Nei’s genetic distance between accessions was 0.3806 with a range of 0.2865 to 0.7091. These results indicated Physalis and tomato had similarity at both molecular marker and DNA sequence levels. Therefore, the molecular markers developed in tomato can be used in genetic study in Physalis. PMID:23166835

Geometry induced sequence of nanoscale Frank–Kasper and quasicrystal mesophases in giant surfactants

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

Yue, Kan; Huang, Mingjun; Marson, Ryan L.

Frank–Kasper (F-K) and quasicrystal phases were originally identified in metal alloys and only sporadically reported in soft materials. These unconventional sphere-packing schemes open up possibilities to design materials with different properties. The challenge in soft materials is how to correlate complex phases built from spheres with the tunable parameters of chemical composition and molecular architecture. Here, we report a complete sequence of various highly ordered mesophases by the self-assembly of specifically designed and synthesized giant surfactants, which are conjugates of hydrophilic polyhedral oligomeric silsesquioxane cages tethered with hydrophobic polystyrene tails. We show that the occurrence of these mesophases results frommore » nanophase separation between the heads and tails and thus is critically dependent on molecular geometry. Variations in molecular geometry achieved by changing the number of tails from one to four not only shift compositional phase boundaries but also stabilize F-K and quasicrystal phases in regions where simple phases of spheroidal micelles are typically observed. These complex self-assembled nanostructures have been identified by combining X-ray scattering techniques and real-space electron microscopy images. Brownian dynamics simulations based on a simplified molecular model confirm the architecture-induced sequence of phases. Our results demonstrate the critical role of molecular architecture in dictating the formation of supramolecular crystals with “soft” spheroidal motifs and provide guidelines to the design of unconventional self-assembled nanostructures.« less

Geometry induced sequence of nanoscale Frank–Kasper and quasicrystal mesophases in giant surfactants

PubMed Central

Yue, Kan; Huang, Mingjun; Marson, Ryan L.; He, Jinlin; Huang, Jiahao; Zhou, Zhe; Wang, Jing; Liu, Chang; Yan, Xuesheng; Wu, Kan; Guo, Zaihong; Liu, Hao; Ni, Peihong; Wesdemiotis, Chrys; Zhang, Wen-Bin; Glotzer, Sharon C.; Cheng, Stephen Z. D.

2016-01-01

Frank–Kasper (F-K) and quasicrystal phases were originally identified in metal alloys and only sporadically reported in soft materials. These unconventional sphere-packing schemes open up possibilities to design materials with different properties. The challenge in soft materials is how to correlate complex phases built from spheres with the tunable parameters of chemical composition and molecular architecture. Here, we report a complete sequence of various highly ordered mesophases by the self-assembly of specifically designed and synthesized giant surfactants, which are conjugates of hydrophilic polyhedral oligomeric silsesquioxane cages tethered with hydrophobic polystyrene tails. We show that the occurrence of these mesophases results from nanophase separation between the heads and tails and thus is critically dependent on molecular geometry. Variations in molecular geometry achieved by changing the number of tails from one to four not only shift compositional phase boundaries but also stabilize F-K and quasicrystal phases in regions where simple phases of spheroidal micelles are typically observed. These complex self-assembled nanostructures have been identified by combining X-ray scattering techniques and real-space electron microscopy images. Brownian dynamics simulations based on a simplified molecular model confirm the architecture-induced sequence of phases. Our results demonstrate the critical role of molecular architecture in dictating the formation of supramolecular crystals with “soft” spheroidal motifs and provide guidelines to the design of unconventional self-assembled nanostructures. PMID:27911786

Identification of single-copy orthologous genes between Physalis and Solanum lycopersicum and analysis of genetic diversity in Physalis using molecular markers.

PubMed

Wei, Jingli; Hu, Xiaorong; Yang, Jingjing; Yang, Wencai

2012-01-01

The genus Physalis includes a number of commercially important edible and ornamental species. Its high nutritional value and potential medicinal properties leads to the increased commercial interest in the products of this genus worldwide. However, lack of molecular markers prevents the detailed study of genetics and phylogeny in Physalis, which limits the progress of breeding. In the present study, we compared the DNA sequences between Physalis and tomato, and attempted to analyze genetic diversity in Physalis using tomato markers. Blasting 23180 DNA sequences derived from Physalis against the International Tomato Annotation Group (ITAG) Release2.3 Predicted CDS (SL2.40) discovered 3356 single-copy orthologous genes between them. A total of 38 accessions from at least six species of Physalis were subjected to genetic diversity analysis using 97 tomato markers and 25 SSR markers derived from P. peruviana. Majority (73.2%) of tomato markers could amplify DNA fragments from at least one accession of Physalis. Diversity in Physalis at molecular level was also detected. The average Nei's genetic distance between accessions was 0.3806 with a range of 0.2865 to 0.7091. These results indicated Physalis and tomato had similarity at both molecular marker and DNA sequence levels. Therefore, the molecular markers developed in tomato can be used in genetic study in Physalis.

Sequence-Mandated, Distinct Assembly of Giant Molecules

DOE PAGES

Zhang, Wei; Lu, Xinlin; Mao, Jialin; ...

2017-10-24

Although controlling the primary structure of synthetic polymers is itself a great challenge, the potential of sequence control for tailoring hierarchical structures remains to be exploited, especially in the creation of new and unconventional phases. A series of model amphiphilic chain-like giant molecules was designed and synthesized by interconnecting both hydrophobic and hydrophilic molecular nanoparticles in precisely defined sequence and composition to investigate their sequence-dependent phase structures. Not only compositional variation changed the self-assembled supramolecular phases, but also specific sequences induce unconventional phase formation, including Frank-Kasper phases. The formation mechanism was attributed to the conformational change driven by the collectivemore » hydrogen bonding and the sequence-mandated topology of the molecules. Lastly, these results show that sequence control in synthetic polymers can have a dramatic impact on polymer properties and self-assembly.« less

Sequence-Mandated, Distinct Assembly of Giant Molecules

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

Zhang, Wei; Lu, Xinlin; Mao, Jialin

Although controlling the primary structure of synthetic polymers is itself a great challenge, the potential of sequence control for tailoring hierarchical structures remains to be exploited, especially in the creation of new and unconventional phases. A series of model amphiphilic chain-like giant molecules was designed and synthesized by interconnecting both hydrophobic and hydrophilic molecular nanoparticles in precisely defined sequence and composition to investigate their sequence-dependent phase structures. Not only compositional variation changed the self-assembled supramolecular phases, but also specific sequences induce unconventional phase formation, including Frank-Kasper phases. The formation mechanism was attributed to the conformational change driven by the collectivemore » hydrogen bonding and the sequence-mandated topology of the molecules. Lastly, these results show that sequence control in synthetic polymers can have a dramatic impact on polymer properties and self-assembly.« less

Mechanical features of various silkworm crystalline considering hydration effect via molecular dynamics simulations.

PubMed

Kim, Yoonjung; Lee, Myeongsang; Choi, Hyunsung; Baek, Inchul; Kim, Jae In; Na, Sungsoo

2018-04-01

Silk materials are receiving significant attention as base materials for various functional nanomaterials and nanodevices, due to its exceptionally high mechanical properties, biocompatibility, and degradable characteristics. Although crystalline silk regions are composed of various repetitive motifs with differing amino acid sequences, how the effect of humidity works differently on each of the motifs and their structural characteristics remains unclear. We report molecular dynamics (MD) simulations on various silkworm fibroins composed of major motifs (i.e. (GAGAGS) n , (GAGAGA) n , and (GAGAGY) n ) at varying degrees of hydration, and reveal how each major motifs of silk fibroins change at each degrees of hydration using MD simulations and their structural properties in mechanical perspective via steered molecular dynamics simulations. Our results explain what effects humidity can have on nanoscale materials and devices consisting of crystalline silk materials.

Quantum mechanical calculations related to ionization and charge transfer in DNA

NASA Astrophysics Data System (ADS)

Cauët, E.; Valiev, M.; Weare, J. H.; Liévin, J.

2012-07-01

Ionization and charge migration in DNA play crucial roles in mechanisms of DNA damage caused by ionizing radiation, oxidizing agents and photo-irradiation. Therefore, an evaluation of the ionization properties of the DNA bases is central to the full interpretation and understanding of the elementary reactive processes that occur at the molecular level during the initial exposure and afterwards. Ab initio quantum mechanical (QM) methods have been successful in providing highly accurate evaluations of key parameters, such as ionization energies (IE) of DNA bases. Hence, in this study, we performed high-level QM calculations to characterize the molecular energy levels and potential energy surfaces, which shed light on ionization and charge migration between DNA bases. In particular, we examined the IEs of guanine, the most easily oxidized base, isolated and embedded in base clusters, and investigated the mechanism of charge migration over two and three stacked guanines. The IE of guanine in the human telomere sequence has also been evaluated. We report a simple molecular orbital analysis to explain how modifications in the base sequence are expected to change the efficiency of the sequence as a hole trap. Finally, the application of a hybrid approach combining quantum mechanics with molecular mechanics brings an interesting discussion as to how the native aqueous DNA environment affects the IE threshold of nucleobases.

Computational complexity of algorithms for sequence comparison, short-read assembly and genome alignment.

PubMed

Baichoo, Shakuntala; Ouzounis, Christos A

A multitude of algorithms for sequence comparison, short-read assembly and whole-genome alignment have been developed in the general context of molecular biology, to support technology development for high-throughput sequencing, numerous applications in genome biology and fundamental research on comparative genomics. The computational complexity of these algorithms has been previously reported in original research papers, yet this often neglected property has not been reviewed previously in a systematic manner and for a wider audience. We provide a review of space and time complexity of key sequence analysis algorithms and highlight their properties in a comprehensive manner, in order to identify potential opportunities for further research in algorithm or data structure optimization. The complexity aspect is poised to become pivotal as we will be facing challenges related to the continuous increase of genomic data on unprecedented scales and complexity in the foreseeable future, when robust biological simulation at the cell level and above becomes a reality. Copyright © 2017 Elsevier B.V. All rights reserved.

Insights into the role of protein molecule size and structure on interfacial properties using designed sequences

PubMed Central

Dwyer, Mirjana Dimitrijev; He, Lizhong; James, Michael; Nelson, Andrew; Middelberg, Anton P. J.

2013-01-01

Mixtures of a large, structured protein with a smaller, unstructured component are inherently complex and hard to characterize at interfaces, leading to difficulties in understanding their interfacial behaviours and, therefore, formulation optimization. Here, we investigated interfacial properties of such a mixed system. Simplicity was achieved using designed sequences in which chemical differences had been eliminated to isolate the effect of molecular size and structure, namely a short unstructured peptide (DAMP1) and its longer structured protein concatamer (DAMP4). Interfacial tension measurements suggested that the size and bulk structuring of the larger molecule led to much slower adsorption kinetics. Neutron reflectometry at equilibrium revealed that both molecules adsorbed as a monolayer to the air–water interface (indicating unfolding of DAMP4 to give a chain of four connected DAMP1 molecules), with a concentration ratio equal to that in the bulk. This suggests the overall free energy of adsorption is equal despite differences in size and bulk structure. At small interfacial extensional strains, only molecule packing influenced the stress response. At larger strains, the effect of size became apparent, with DAMP4 registering a higher stress response and interfacial elasticity. When both components were present at the interface, most stress-dissipating movement was achieved by DAMP1. This work thus provides insights into the role of proteins' molecular size and structure on their interfacial properties, and the designed sequences introduced here can serve as effective tools for interfacial studies of proteins and polymers. PMID:23303222

Unique Conformation in a Natural Interruption Sequence of Type XIX Collagen Revealed by Its High-Resolution Crystal Structure.

PubMed

Xu, Tingting; Zhou, Cong-Zhao; Xiao, Jianxi; Liu, Jinsong

2018-02-20

Naturally occurring interruptions in nonfibrillar collagen play key roles in molecular flexibility, collagen degradation, and ligand binding. The structural feature of the interruption sequences and the molecular basis for their functions have not been well studied. Here, we focused on a G5G type natural interruption sequence G-POALO-G from human type XIX collagen, a homotrimer collagen, as this sequence possesses distinct properties compared with those of a pathological similar Gly mutation sequence in collagen mimic peptides. We determined the crystal structures of the host-guest peptide (GPO) 3 -GPOALO-(GPO) 4 to 1.03 Å resolution in two crystal forms. In these structures, the interruption zone brings localized disruptions to the triple helix and introduces a light 6-8° bend with the same directional preference to the whole molecule, which may correspond structurally to the first physiological kink site in type XIX collagen. Furthermore, at the G5G interruption site, the presence of Ala and Leu residues, both with free N-H groups, allows the formation of more direct and water-mediated interchain hydrogen bonds than in the related Gly → Ala structure. These could partly explain the difference in thermal stability between the different interruptions. In addition, our structures provide a detailed view of the dynamic property of such an interrupted zone with respect to hydrogen bonding topology, torsion angles, and helical parameters. Our results, for the first time, also identified the binding of zinc to the end of the triple helix. These findings will shed light on how the interruption sequence influences the conformation of the collagen molecule and provide a structural basis for further functional studies.

Stellar and Circumstellar Properties of the Pre-Main-Sequence Binary GV Tau from Infrared Spectroscopy

NASA Astrophysics Data System (ADS)

Doppmann, Greg W.; Najita, Joan R.; Carr, John S.

2008-09-01

We report spatially resolved spectroscopy of both components of the low-mass pre-main-sequence binary GV Tau. High-resolution spectroscopy in the K and L bands is used to characterize the stellar properties of the binary and to explore the nature of the circumstellar environment. We find that the southern component, GV Tau S, is a radial velocity variable, possibly as a result of an unseen low-mass companion. The strong warm gaseous HCN absorption reported previously by Gibb and coworkers toward GV Tau S was not present during the epoch of our observations. Instead, we detect warm (~500 K) molecular absorption with similar properties toward the northern infrared companion, GV Tau N. At the epoch of our observations, the absorbing gas toward GV Tau N was approximately at the radial velocity of the GV Tau molecular envelope, but it was redshifted with respect to the star by ~13 km s-1. One interpretation of our results is that GV Tau N is also a binary and that most of the warm molecular absorption arises in a circumbinary disk viewed close to edge-on. Data presented herein were obtained at the W. M. Keck Observatory from telescope time allocated to the National Aeronautics and Space Administration through the agency's scientific partnership with the California Institute of Technology and the University of California. The Observatory was made possible by the generous financial support of the W. M. Keck Foundation.

Fuzzy cluster analysis of simple physicochemical properties of amino acids for recognizing secondary structure in proteins.

PubMed Central

Mocz, G.

1995-01-01

Fuzzy cluster analysis has been applied to the 20 amino acids by using 65 physicochemical properties as a basis for classification. The clustering products, the fuzzy sets (i.e., classical sets with associated membership functions), have provided a new measure of amino acid similarities for use in protein folding studies. This work demonstrates that fuzzy sets of simple molecular attributes, when assigned to amino acid residues in a protein's sequence, can predict the secondary structure of the sequence with reasonable accuracy. An approach is presented for discriminating standard folding states, using near-optimum information splitting in half-overlapping segments of the sequence of assigned membership functions. The method is applied to a nonredundant set of 252 proteins and yields approximately 73% matching for correctly predicted and correctly rejected residues with approximately 60% overall success rate for the correctly recognized ones in three folding states: alpha-helix, beta-strand, and coil. The most useful attributes for discriminating these states appear to be related to size, polarity, and thermodynamic factors. Van der Waals volume, apparent average thickness of surrounding molecular free volume, and a measure of dimensionless surface electron density can explain approximately 95% of prediction results. hydrogen bonding and hydrophobicity induces do not yet enable clear clustering and prediction. PMID:7549882

Overcoming Ehrlich-Schwöbel barrier in (1 1 1)A GaAs molecular beam epitaxy

NASA Astrophysics Data System (ADS)

Ritzmann, Julian; Schott, Rüdiger; Gross, Katherine; Reuter, Dirk; Ludwig, Arne; Wieck, Andreas D.

2018-01-01

In this work, we first study the effect of different growth parameters on the molecular beam epitaxy (MBE) growth of GaAs layers on (1 1 1)A oriented substrates. After that we present a method for the MBE growth of atomically smooth layers by sequences of growth and annealing phases. The samples exhibit low surface roughness and good electrical properties shown by atomic force microscopy (AFM), scanning electron microscopy (SEM) and van-der-Pauw Hall measurements.

Predicting RNA Duplex Dimerization Free-Energy Changes upon Mutations Using Molecular Dynamics Simulations.

PubMed

Sakuraba, Shun; Asai, Kiyoshi; Kameda, Tomoshi

2015-11-05

The dimerization free energies of RNA-RNA duplexes are fundamental values that represent the structural stability of RNA complexes. We report a comparative analysis of RNA-RNA duplex dimerization free-energy changes upon mutations, estimated from a molecular dynamics simulation and experiments. A linear regression for nine pairs of double-stranded RNA sequences, six base pairs each, yielded a mean absolute deviation of 0.55 kcal/mol and an R(2) value of 0.97, indicating quantitative agreement between simulations and experimental data. The observed accuracy indicates that the molecular dynamics simulation with the current molecular force field is capable of estimating the thermodynamic properties of RNA molecules.

Molecular Properties of Poliovirus Isolates: Nucleotide Sequence Analysis, Typing by PCR and Real-Time RT-PCR.

PubMed

Burns, Cara C; Kilpatrick, David R; Iber, Jane C; Chen, Qi; Kew, Olen M

2016-01-01

Virologic surveillance is essential to the success of the World Health Organization initiative to eradicate poliomyelitis. Molecular methods have been used to detect polioviruses in tissue culture isolates derived from stool samples obtained through surveillance for acute flaccid paralysis. This chapter describes the use of realtime PCR assays to identify and serotype polioviruses. In particular, a degenerate, inosine-containing, panpoliovirus (panPV) PCR primer set is used to distinguish polioviruses from NPEVs. The high degree of nucleotide sequence diversity among polioviruses presents a challenge to the systematic design of nucleic acid-based reagents. To accommodate the wide variability and rapid evolution of poliovirus genomes, degenerate codon positions on the template were matched to mixed-base or deoxyinosine residues on both the primers and the TaqMan™ probes. Additional assays distinguish between Sabin vaccine strains and non-Sabin strains. This chapter also describes the use of generic poliovirus specific primers, along with degenerate and inosine-containing primers, for routine VP1 sequencing of poliovirus isolates. These primers, along with nondegenerate serotype-specific Sabin primers, can also be used to sequence individual polioviruses in mixtures.

Molecular coevolution of mammalian ribosomal gene terminator sequences and the transcription termination factor TTF-I.

PubMed Central

Evers, R; Grummt, I

1995-01-01

Both the DNA elements and the nuclear factors that direct termination of ribosomal gene transcription exhibit species-specific differences. Even between mammals--e.g., human and mouse--the termination signals are not identical and the respective transcription termination factors (TTFs) which bind to the terminator sequence are not fully interchangeable. To elucidate the molecular basis for this species-specificity, we have cloned TTF-I from human and mouse cells and compared their structural and functional properties. Recombinant TTF-I exhibits species-specific DNA binding and terminates transcription both in cell-free transcription assays and in transfection experiments. Chimeric constructs of mouse TTF-I and human TTF-I reveal that the major determinant for species-specific DNA binding resides within the C terminus of TTF-I. Replacing 31 C-terminal amino acids of mouse TTF-I with the homologous human sequences relaxes the DNA-binding specificity and, as a consequence, allows the chimeric factor to bind the human terminator sequence and to specifically stop rDNA transcription. Images Fig. 2 Fig. 3 Fig. 4 PMID:7597036

Sequence-structure correlations in silk: Poly-Ala repeat of N. clavipes MaSp1 is naturally optimized at a critical length scale.

PubMed

Bratzel, Graham; Buehler, Markus J

2012-03-01

Spider silk is a self-assembling biopolymer that outperforms many known materials in terms of its mechanical performance despite being constructed from simple and inferior building blocks. While experimental studies have shown that the molecular structure of silk has a direct influence on the stiffness, toughness, and failure strength of silk, few molecular-level analyses of the nanostructure of silk assemblies in particular under variations of genetic sequences have been reported. Here we report atomistic-level structures of the MaSp1 protein from the Nephila Clavipes spider dragline silk sequence, obtained using an in silico approach based on replica exchange molecular dynamics (REMD) and explicit water molecular dynamics. We apply this method to study the effects of a systematic variation of the poly-alanine repeat lengths, a parameter controlled by the genetic makeup of silk, on the resulting molecular structure of silk at the nanoscale. Confirming earlier experimental and computational work, a structural analysis reveals that poly-alanine regions in silk predominantly form distinct and orderly β-sheet crystal domains while disorderly regions are formed by glycine-rich repeats that consist of 3(10)-helix type structures and β-turns. Our predictions are directly validated against experimental data based on dihedral angle pair calculations presented in Ramachandran plots combined with an analysis of the secondary structure content. The key result of our study is our finding of a strong dependence of the resulting silk nanostructure depending on the poly-alanine length. We observe that the wildtype poly-alanine repeat length of six residues defines a critical minimum length that consistently results in clearly defined β-sheet nanocrystals. For poly-alanine lengths below six, the β-sheet nanocrystals are not well-defined or not visible at all, while for poly-alanine lengths at and above six, the characteristic nanocomposite structure of silk emerges with no significant improvement of the quality of the β-sheet nanocrystal geometry. We present a simple biophysical model that explains these computational observations based on the mechanistic insight gained from the molecular simulations. Our findings set the stage for understanding how variations in the spidroin sequence can be used to engineer the structure and thereby functional properties of this biological superfiber, and present a design strategy for the genetic optimization of spidroins for enhanced mechanical properties. The approach used here may also find application in the design of other self-assembled molecular structures and fibers and in particular biologically inspired or completely synthetic systems. Copyright © 2011 Elsevier Ltd. All rights reserved.

Bacterial CRISPR Regions: General Features and their Potential for Epidemiological Molecular Typing Studies.

PubMed

Karimi, Zahra; Ahmadi, Ali; Najafi, Ali; Ranjbar, Reza

2018-01-01

CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) loci as novel and applicable regions in prokaryotic genomes have gained great attraction in the post genomics era. These unique regions are diverse in number and sequence composition in different pathogenic bacteria and thereby can be a suitable candidate for molecular epidemiology and genotyping studies. Results:Furthermore, the arrayed structure of CRISPR loci (several unique repeats spaced with the variable sequence) and associated cas genes act as an active prokaryotic immune system against viral replication and conjugative elements. This property can be used as a tool for RNA editing in bioengineering studies. The aim of this review was to survey some details about the history, nature, and potential applications of CRISPR arrays in both genetic engineering and bacterial genotyping studies.

Molecular dynamics studies of the 3D structure and planar ligand binding of a quadruplex dimer.

PubMed

Li, Ming-Hui; Luo, Quan; Xue, Xiang-Gui; Li, Ze-Sheng

2011-03-01

G-rich sequences can fold into a four-stranded structure called a G-quadruplex, and sequences with short loops are able to aggregate to form stable quadruplex multimers. Few studies have characterized the properties of this variety of quadruplex multimers. Using molecular modeling and molecular dynamics simulations, the present study investigated a dimeric G-quadruplex structure formed from a simple sequence of d(GGGTGGGTGGGTGGGT) (G1), and its interactions with a planar ligand of a perylene derivative (Tel03). A series of analytical methods, including free energy calculations and principal components analysis (PCA), was used. The results show that a dimer structure with stacked parallel monomer structures is maintained well during the entire simulation. Tel03 can bind to the dimer efficiently through end stacking, and the binding mode of the ligand stacked with the 3'-terminal thymine base is most favorable. PCA showed that the dominant motions in the free dimer occur on the loop regions, and the presence of the ligand reduces the flexibility of the loops. Our investigation will assist in understanding the geometric structure of stacked G-quadruplex multimers and may be helpful as a platform for rational drug design.

Ligninolytic peroxidase genes in the oyster mushroom genome: heterologous expression, molecular structure, catalytic and stability properties, and lignin-degrading ability

Treesearch

Elena Fernández-Fueyo; Francisco J Ruiz-Dueñas; María Jesús Martinez; Antonio Romero; Kenneth E Hammel; Francisco Javier Medrano; Angel T. Martínez

2014-01-01

Background: The genome of Pleurotus ostreatus, an important edible mushroom and a model ligninolytic organism of interest in lignocellulose biorefineries due to its ability to delignify agricultural wastes, was sequenced with the purpose of identifying and characterizing the enzymes responsible for lignin degradation. ...

Purification, characterization and sequence analysis of Omp50,a new porin isolated from Campylobacter jejuni.

PubMed Central

Bolla, J M; Dé, E; Dorez, A; Pagès, J M

2000-01-01

A novel pore-forming protein identified in Campylobacter was purified by ion-exchange chromatography and named Omp50 according to both its molecular mass and its outer membrane localization. We observed a pore-forming ability of Omp50 after re-incorporation into artificial membranes. The protein induced cation-selective channels with major conductance values of 50-60 pS in 1 M NaCl. N-terminal sequencing allowed us to identify the predicted coding sequence Cj1170c from the Campylobacter jejuni genome database as the corresponding gene in the NCTC 11168 genome sequence. The gene, designated omp50, consists of a 1425 bp open reading frame encoding a deduced 453-amino acid protein with a calculated pI of 5.81 and a molecular mass of 51169.2 Da. The protein possessed a 20-amino acid leader sequence. No significant similarity was found between Omp50 and porin protein sequences already determined. Moreover, the protein showed only weak sequence identity with the major outer-membrane protein (MOMP) of Campylobacter, correlating with the absence of antigenic cross-reactivity between these two proteins. Omp50 is expressed in C. jejuni and Campylobacter lari but not in Campylobacter coli. The gene, however, was detected in all three species by PCR. According to its conformation and functional properties, the protein would belong to the family of outer-membrane monomeric porins. PMID:11104668

Mapping the pericentric heterochromatin by comparative genomic hybridization analysis and chromosome deletions in Drosophila melanogaster

PubMed Central

He, Bing; Caudy, Amy; Parsons, Lance; Rosebrock, Adam; Pane, Attilio; Raj, Sandeep; Wieschaus, Eric

2012-01-01

Heterochromatin represents a significant portion of eukaryotic genomes and has essential structural and regulatory functions. Its molecular organization is largely unknown due to difficulties in sequencing through and assembling repetitive sequences enriched in the heterochromatin. Here we developed a novel strategy using chromosomal rearrangements and embryonic phenotypes to position unmapped Drosophila melanogaster heterochromatic sequence to specific chromosomal regions. By excluding sequences that can be mapped to the assembled euchromatic arms, we identified sequences that are specific to heterochromatin and used them to design heterochromatin specific probes (“H-probes”) for microarray. By comparative genomic hybridization (CGH) analyses of embryos deficient for each chromosome or chromosome arm, we were able to map most of our H-probes to specific chromosome arms. We also positioned sequences mapped to the second and X chromosomes to finer intervals by analyzing smaller deletions with breakpoints in heterochromatin. Using this approach, we were able to map >40% (13.9 Mb) of the previously unmapped heterochromatin sequences assembled by the whole-genome sequencing effort on arm U and arm Uextra to specific locations. We also identified and mapped 110 kb of novel heterochromatic sequences. Subsequent analyses revealed that sequences located within different heterochromatic regions have distinct properties, such as sequence composition, degree of repetitiveness, and level of underreplication in polytenized tissues. Surprisingly, although heterochromatin is generally considered to be transcriptionally silent, we detected region-specific temporal patterns of transcription in heterochromatin during oogenesis and early embryonic development. Our study provides a useful approach to elucidate the molecular organization and function of heterochromatin and reveals region-specific variation of heterochromatin. PMID:22745230

Substantial reservoirs of molecular hydrogen in the debris disks around young stars.

PubMed

Thi, W F; Blake, G A; van Dishoeck, E F; van Zadelhoff, G J; Horn, J M; Becklin, E E; Mannings, V; Sargent, A I; van Den Ancker, M E; Natta, A

2001-01-04

Circumstellar accretion disks transfer matter from molecular clouds to young stars and to the sites of planet formation. The disks observed around pre-main-sequence stars have properties consistent with those expected for the pre-solar nebula from which our own Solar System formed 4.5 Gyr ago. But the 'debris' disks that encircle more than 15% of nearby main-sequence stars appear to have very small amounts of gas, based on observations of the tracer molecule carbon monoxide: these observations have yielded gas/dust ratios much less than 0.1, whereas the interstellar value is about 100 (ref. 9). Here we report observations of the lowest rotational transitions of molecular hydrogen (H2) that reveal large quantities of gas in the debris disks around the stars beta Pictoris, 49 Ceti and HD135344. The gas masses calculated from the data are several hundreds to a thousand times greater than those estimated from the CO observations, and yield gas/dust ratios of the same order as the interstellar value.

Purification and sequence analysis of 4-methyl-5-nitrocatechol oxygenase from Burkholderia sp. strain DNT.

PubMed Central

Haigler, B E; Suen, W C; Spain, J C

1996-01-01

4-Methyl-5-nitrocatechol (MNC) is an intermediate in the degradation of 2,4-dinitrotoluene by Burkholderia sp. strain DNT. In the presence of NADPH and oxygen, MNC monooxygenase catalyzes the removal of the nitro group from MNC to form 2-hydroxy-5-methylquinone. The gene (dntB) encoding MNC monooxygenase has been previously cloned and characterized. In order to examine the properties of MNC monooxygenase and to compare it with other enzymes, we sequenced the gene encoding the MNC monooxygenase and purified the enzyme from strain DNT. dntB was localized within a 2.2-kb ApaI DNA fragment. Sequence analysis of this fragment revealed an open reading frame of 1,644 bp with an N-terminal amino acid sequence identical to that of purified MNC monooxygenase from strain DNT. Comparison of the derived amino acid sequences with those of other genes showed that DntB contains the highly conserved ADP and flavin adenine dinucleotide (FAD) binding motifs characteristic of flavoprotein hydroxylases. MNC monooxygenase was purified to homogeneity from strain DNT by anion exchange and gel filtration chromatography. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed a single protein with a molecular weight of 60,200, which is consistent with the size determined from the gene sequence. The native molecular weight determined by gel filtration was 65,000, which indicates that the native enzyme is a monomer. It used either NADH or NADPH as electron donors, and NADPH was the preferred cofactor. The purified enzyme contained 1 mol of FAD per mol of protein, which is also consistent with the detection of an FAD binding motif in the amino acid sequence of DntB. MNC monooxygenase has a narrow substrate specificity. MNC and 4-nitrocatechol are good substrates whereas 3-methyl-4-nitrophenol, 3-methyl-4-nitrocatechol, 4-nitrophenol, 3-nitrophenol, and 4-chlorocatechol were not. These studies suggest that MNC monooxygenase is a flavoprotein that shares some properties with previously studied nitrophenol oxygenases. PMID:8830701

Molecular characterization of a mannoprotein with homology to chitin deacetylases that stimulates T cell responses to Cryptococcus neoformans.

PubMed

Levitz, S M; Nong , S; Mansour, M K; Huang, C; Specht, C A

2001-08-28

The fungus Cryptococcus neoformans is a major cause of morbidity and mortality in patients with impaired CD4(+) T cell function, particularly those with AIDS. To identify cryptococcal antigens that could serve as vaccine candidates by stimulating T cell responses, C. neoformans-reactive CD4(+) T cell hybridomas were generated by immunization of C57BL/6 mice and fusion of splenocytes with thymoma cells. The antigen that stimulated one of the hybridomas, designated P1D6, to produce IL-2 was purified to homogeneity by sequential anion exchange chromatography, hydrophobic interaction chromatography, and SDS/PAGE. Based on its apparent molecular mass of 98 kDa and mannosylation, the antigen of interest was named MP98. MP98 was N terminal-sequenced, and the gene encoding the protein was cloned and sequenced. Recombinant MP98, expressed in Saccharomyces cerevisiae, stimulated P1D6 to produce IL-2. Analysis of the derived 458-aa sequence of MP98 reveals an N-terminal cleavable signal sequence, a polysaccharide deacetylase domain found in fungal chitin deacetylases, and a serine/threonine-rich C-terminal region. Overall, there were 103 serine/threonine residues serving as potential O-linked glycosylation sites as well as 12 possible N-linked glycosylation sites. Thus, a C. neoformans mannoprotein has been characterized that stimulates T cell responses and has molecular properties of a chitin deacetylase.

Comparative Genomics of Bacillus species and its Relevance in Industrial Microbiology.

PubMed

Sharma, Archana; Satyanarayana, T

2013-01-01

With the advent of high throughput sequencing platforms and relevant analytical tools, the rate of microbial genome sequencing has accelerated which has in turn led to better understanding of microbial molecular biology and genetics. The complete genome sequences of important industrial organisms provide opportunities for human health, industry, and the environment. Bacillus species are the dominant workhorses in industrial fermentations. Today, genome sequences of several Bacillus species are available, and comparative genomics of this genus helps in understanding their physiology, biochemistry, and genetics. The genomes of these bacterial species are the sources of many industrially important enzymes and antibiotics and, therefore, provide an opportunity to tailor enzymes with desired properties to suit a wide range of applications. A comparative account of strengths and weaknesses of the different sequencing platforms are also highlighted in the review.

Nanoparticles engineered to bind cellular motors for efficient delivery.

PubMed

Dalmau-Mena, Inmaculada; Del Pino, Pablo; Pelaz, Beatriz; Cuesta-Geijo, Miguel Ángel; Galindo, Inmaculada; Moros, María; de la Fuente, Jesús M; Alonso, Covadonga

2018-03-30

Dynein is a cytoskeletal molecular motor protein that transports cellular cargoes along microtubules. Biomimetic synthetic peptides designed to bind dynein have been shown to acquire dynamic properties such as cell accumulation and active intra- and inter-cellular motion through cell-to-cell contacts and projections to distant cells. On the basis of these properties dynein-binding peptides could be used to functionalize nanoparticles for drug delivery applications. Here, we show that gold nanoparticles modified with dynein-binding delivery sequences become mobile, powered by molecular motor proteins. Modified nanoparticles showed dynamic properties, such as travelling the cytosol, crossing intracellular barriers and shuttling the nuclear membrane. Furthermore, nanoparticles were transported from one cell to another through cell-to-cell contacts and quickly spread to distant cells through cell projections. The capacity of these motor-bound nanoparticles to spread to many cells and increasing cellular retention, thus avoiding losses and allowing lower dosage, could make them candidate carriers for drug delivery.

Hydration properties of natural and synthetic DNA sequences with methylated adenine or cytosine bases in the R.DpnI target and BDNF promoter studied by molecular dynamics simulations

NASA Astrophysics Data System (ADS)

Shanak, Siba; Helms, Volkhard

2014-12-01

Adenine and cytosine methylation are two important epigenetic modifications of DNA sequences at the levels of the genome and transcriptome. To characterize the differential roles of methylating adenine or cytosine with respect to their hydration properties, we performed conventional MD simulations and free energy perturbation calculations for two particular DNA sequences, namely the brain-derived neurotrophic factor (BDNF) promoter and the R.DpnI-bound DNA that are known to undergo methylation of C5-methyl cytosine and N6-methyl adenine, respectively. We found that a single methylated cytosine has a clearly favorable hydration free energy over cytosine since the attached methyl group has a slightly polar character. In contrast, capping the strongly polar N6 of adenine with a methyl group gives a slightly unfavorable contribution to its free energy of solvation. Performing the same demethylation in the context of a DNA double-strand gave quite similar results for the more solvent-accessible cytosine but much more unfavorable results for the rather buried adenine. Interestingly, the same demethylation reactions are far more unfavorable when performed in the context of the opposite (BDNF or R.DpnI target) sequence. This suggests a natural preference for methylation in a specific sequence context. In addition, free energy calculations for demethylating adenine or cytosine in the context of B-DNA vs. Z-DNA suggest that the conformational B-Z transition of DNA transition is rather a property of cytosine methylated sequences but is not preferable for the adenine-methylated sequences investigated here.

Hydration properties of natural and synthetic DNA sequences with methylated adenine or cytosine bases in the R.DpnI target and BDNF promoter studied by molecular dynamics simulations.

PubMed

Shanak, Siba; Helms, Volkhard

2014-12-14

Adenine and cytosine methylation are two important epigenetic modifications of DNA sequences at the levels of the genome and transcriptome. To characterize the differential roles of methylating adenine or cytosine with respect to their hydration properties, we performed conventional MD simulations and free energy perturbation calculations for two particular DNA sequences, namely the brain-derived neurotrophic factor (BDNF) promoter and the R.DpnI-bound DNA that are known to undergo methylation of C5-methyl cytosine and N6-methyl adenine, respectively. We found that a single methylated cytosine has a clearly favorable hydration free energy over cytosine since the attached methyl group has a slightly polar character. In contrast, capping the strongly polar N6 of adenine with a methyl group gives a slightly unfavorable contribution to its free energy of solvation. Performing the same demethylation in the context of a DNA double-strand gave quite similar results for the more solvent-accessible cytosine but much more unfavorable results for the rather buried adenine. Interestingly, the same demethylation reactions are far more unfavorable when performed in the context of the opposite (BDNF or R.DpnI target) sequence. This suggests a natural preference for methylation in a specific sequence context. In addition, free energy calculations for demethylating adenine or cytosine in the context of B-DNA vs. Z-DNA suggest that the conformational B-Z transition of DNA transition is rather a property of cytosine methylated sequences but is not preferable for the adenine-methylated sequences investigated here.

Soft hydrogel materials from elastomeric gluten-mimetic proteins

NASA Astrophysics Data System (ADS)

Bagheri, Mehran; Scott, Shane; Wan, Fan; Dick, Scott; Harden, James; Biomolecular Assemblies Team

2014-03-01

Elastomeric proteins are ubiquitous in both animal and plant tissues, where they are responsible for the elastic response and mechanical resilience of tissues. In addition to fundamental interest in the molecular origins of their elastic behaviour, this class of proteins has great potential for use in biomaterial applications. The structural and elastomeric properties of these proteins are thought to be controlled by a subtle balance between hydrophobic interactions and entropic effects, and in many cases their characteristic properties can be recapitulated by multi-block protein polymers formed from repeats of short, characteristic polypeptide motifs. We have developed biomimetic multi-block protein polymers based on variants of several elastomeric gluten consensus sequences. These proteins include constituents designed to maximize their solubility in aqueous solution and minimize the formation of extended secondary structure. Thus, they are examples of elastic intrinsically disordered proteins. In addition, the proteins have distributed tyrosine residues which allow for inter-molecular crosslinking to form hydrogel networks. In this talk, we present experimental and simulation studies of the molecular and materials properties of these proteins and their assemblies.

Blends of cysteine-containing proteins

NASA Astrophysics Data System (ADS)

Barone, Justin

2005-03-01

Many agricultural wastes are made of proteins such as keratin, lactalbumin, gluten, and albumin. These proteins contain the amino acid cysteine. Cysteine allows for the formation of inter-and intra-molecular sulfur-sulfur bonds. Correlations are made between the properties of films made from the proteins and the amino acid sequence. Blends of cysteine-containing proteins show possible synergies in physical properties at intermediate concentrations. FT-IR spectroscopy shows increased hydrogen bonding at intermediate concentrations suggesting that this contributes to increased physical properties. DSC shows limited miscibility and the formation of new crystalline phases in the blends suggesting that this too contributes.

Rediscovering Medicinal Plants' Potential with OMICS: Microsatellite Survey in Expressed Sequence Tags of Eleven Traditional Plants with Potent Antidiabetic Properties

PubMed Central

Sahu, Jagajjit; Sen, Priyabrata; Choudhury, Manabendra Dutta; Dehury, Budheswar; Barooah, Madhumita; Modi, Mahendra Kumar

2014-01-01

Abstract Herbal medicines and traditionally used medicinal plants present an untapped potential for novel molecular target discovery using systems science and OMICS biotechnology driven strategies. Since up to 40% of the world's poor people have no access to government health services, traditional and folk medicines are often the only therapeutics available to them. In this vein, North East (NE) India is recognized for its rich bioresources. As part of the Indo-Burma hotspot, it is regarded as an epicenter of biodiversity for several plants having myriad traditional uses, including medicinal use. However, the improvement of these valuable bioresources through molecular breeding strategies, for example, using genic microsatellites or Simple Sequence Repeats (SSRs) or Expressed Sequence Tags (ESTs)-derived SSRs has not been fully utilized in large scale to date. In this study, we identified a total of 47,700 microsatellites from 109,609 ESTs of 11 medicinal plants (pineapple, papaya, noyontara, bitter orange, bermuda brass, ratalu, barbados nut, mango, mulberry, lotus, and guduchi) having proven antidiabetic properties. A total of 58,159 primer pairs were designed for the non-redundant 8060 SSR-positive ESTs and putative functions were assigned to 4483 unique contigs. Among the identified microsatellites, excluding mononucleotide repeats, di-/trinucleotides are predominant, among which repeat motifs of AG/CT and AAG/CTT were most abundant. Similarity search of SSR containing ESTs and antidiabetic gene sequences revealed 11 microsatellites linked to antidiabetic genes in five plants. GO term enrichment analysis revealed a total of 80 enriched GO terms widely distributed in 53 biological processes, 17 molecular functions, and 10 cellular components associated with the 11 markers. The present study therefore provides concrete insights into the frequency and distribution of SSRs in important medicinal resources. The microsatellite markers reported here markedly add to the genetic stock for cross transferability in these plants and the literature on biomarkers and novel drug discovery for common chronic diseases such as diabetes. PMID:24802971

Rediscovering medicinal plants' potential with OMICS: microsatellite survey in expressed sequence tags of eleven traditional plants with potent antidiabetic properties.

PubMed

Sahu, Jagajjit; Sen, Priyabrata; Choudhury, Manabendra Dutta; Dehury, Budheswar; Barooah, Madhumita; Modi, Mahendra Kumar; Talukdar, Anupam Das

2014-05-01

Herbal medicines and traditionally used medicinal plants present an untapped potential for novel molecular target discovery using systems science and OMICS biotechnology driven strategies. Since up to 40% of the world's poor people have no access to government health services, traditional and folk medicines are often the only therapeutics available to them. In this vein, North East (NE) India is recognized for its rich bioresources. As part of the Indo-Burma hotspot, it is regarded as an epicenter of biodiversity for several plants having myriad traditional uses, including medicinal use. However, the improvement of these valuable bioresources through molecular breeding strategies, for example, using genic microsatellites or Simple Sequence Repeats (SSRs) or Expressed Sequence Tags (ESTs)-derived SSRs has not been fully utilized in large scale to date. In this study, we identified a total of 47,700 microsatellites from 109,609 ESTs of 11 medicinal plants (pineapple, papaya, noyontara, bitter orange, bermuda brass, ratalu, barbados nut, mango, mulberry, lotus, and guduchi) having proven antidiabetic properties. A total of 58,159 primer pairs were designed for the non-redundant 8060 SSR-positive ESTs and putative functions were assigned to 4483 unique contigs. Among the identified microsatellites, excluding mononucleotide repeats, di-/trinucleotides are predominant, among which repeat motifs of AG/CT and AAG/CTT were most abundant. Similarity search of SSR containing ESTs and antidiabetic gene sequences revealed 11 microsatellites linked to antidiabetic genes in five plants. GO term enrichment analysis revealed a total of 80 enriched GO terms widely distributed in 53 biological processes, 17 molecular functions, and 10 cellular components associated with the 11 markers. The present study therefore provides concrete insights into the frequency and distribution of SSRs in important medicinal resources. The microsatellite markers reported here markedly add to the genetic stock for cross transferability in these plants and the literature on biomarkers and novel drug discovery for common chronic diseases such as diabetes.

An organismic critique of molecular darwinism.

PubMed

Wicken, J S

1985-12-21

The molecular darwinian approach to the emergence of life treats the competition between RNA sequences for nucleotide resources as the primordial selective process in prebiotic evolution, which prescribes possible pathways for the subsequent elaboration of organizational relationships. Since success in this competition is determined by the "phenotypic" properties of RNA strands in the absence of organizational context, the genesis of biotic organization is dependent upon the establishment of co-operative, hypercyclic interactions between competing RNA sequences. The thesis of this paper is that hypercycle theory is based on unwarranted assumptions about the conditions of prebiotic evolution, and that the implications of these assumptions run counter to both empirical evidence and to the rational by which natural selection operates in evolution generally. An organismic alternative to hypercycle theory is suggested, based on the catalytic microsphere and the thermodynamics of selection.

Bacterial CRISPR Regions: General Features and their Potential for Epidemiological Molecular Typing Studies

PubMed Central

Karimi, Zahra; Ahmadi, Ali; Najafi, Ali; Ranjbar, Reza

2018-01-01

Introduction: CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) loci as novel and applicable regions in prokaryotic genomes have gained great attraction in the post genomics era. Methods: These unique regions are diverse in number and sequence composition in different pathogenic bacteria and thereby can be a suitable candidate for molecular epidemiology and genotyping studies. Results:Furthermore, the arrayed structure of CRISPR loci (several unique repeats spaced with the variable sequence) and associated cas genes act as an active prokaryotic immune system against viral replication and conjugative elements. This property can be used as a tool for RNA editing in bioengineering studies. Conclusion: The aim of this review was to survey some details about the history, nature, and potential applications of CRISPR arrays in both genetic engineering and bacterial genotyping studies. PMID:29755603

Bio-AIMS Collection of Chemoinformatics Web Tools based on Molecular Graph Information and Artificial Intelligence Models.

PubMed

Munteanu, Cristian R; Gonzalez-Diaz, Humberto; Garcia, Rafael; Loza, Mabel; Pazos, Alejandro

2015-01-01

The molecular information encoding into molecular descriptors is the first step into in silico Chemoinformatics methods in Drug Design. The Machine Learning methods are a complex solution to find prediction models for specific biological properties of molecules. These models connect the molecular structure information such as atom connectivity (molecular graphs) or physical-chemical properties of an atom/group of atoms to the molecular activity (Quantitative Structure - Activity Relationship, QSAR). Due to the complexity of the proteins, the prediction of their activity is a complicated task and the interpretation of the models is more difficult. The current review presents a series of 11 prediction models for proteins, implemented as free Web tools on an Artificial Intelligence Model Server in Biosciences, Bio-AIMS (http://bio-aims.udc.es/TargetPred.php). Six tools predict protein activity, two models evaluate drug - protein target interactions and the other three calculate protein - protein interactions. The input information is based on the protein 3D structure for nine models, 1D peptide amino acid sequence for three tools and drug SMILES formulas for two servers. The molecular graph descriptor-based Machine Learning models could be useful tools for in silico screening of new peptides/proteins as future drug targets for specific treatments.

Sequence-Dependent Structure/Function Relationships of Catalytic Peptide-Enabled Gold Nanoparticles Generated under Ambient Synthetic Conditions.

PubMed

Bedford, Nicholas M; Hughes, Zak E; Tang, Zhenghua; Li, Yue; Briggs, Beverly D; Ren, Yang; Swihart, Mark T; Petkov, Valeri G; Naik, Rajesh R; Knecht, Marc R; Walsh, Tiffany R

2016-01-20

Peptide-enabled nanoparticle (NP) synthesis routes can create and/or assemble functional nanomaterials under environmentally friendly conditions, with properties dictated by complex interactions at the biotic/abiotic interface. Manipulation of this interface through sequence modification can provide the capability for material properties to be tailored to create enhanced materials for energy, catalysis, and sensing applications. Fully realizing the potential of these materials requires a comprehensive understanding of sequence-dependent structure/function relationships that is presently lacking. In this work, the atomic-scale structures of a series of peptide-capped Au NPs are determined using a combination of atomic pair distribution function analysis of high-energy X-ray diffraction data and advanced molecular dynamics (MD) simulations. The Au NPs produced with different peptide sequences exhibit varying degrees of catalytic activity for the exemplar reaction 4-nitrophenol reduction. The experimentally derived atomic-scale NP configurations reveal sequence-dependent differences in structural order at the NP surface. Replica exchange with solute-tempering MD simulations are then used to predict the morphology of the peptide overlayer on these Au NPs and identify factors determining the structure/catalytic properties relationship. We show that the amount of exposed Au surface, the underlying surface structural disorder, and the interaction strength of the peptide with the Au surface all influence catalytic performance. A simplified computational prediction of catalytic performance is developed that can potentially serve as a screening tool for future studies. Our approach provides a platform for broadening the analysis of catalytic peptide-enabled metallic NP systems, potentially allowing for the development of rational design rules for property enhancement.

A systematic molecular dynamics study of nearest-neighbor effects on base pair and base pair step conformations and fluctuations in B-DNA

PubMed Central

Lavery, Richard; Zakrzewska, Krystyna; Beveridge, David; Bishop, Thomas C.; Case, David A.; Cheatham, Thomas; Dixit, Surjit; Jayaram, B.; Lankas, Filip; Laughton, Charles; Maddocks, John H.; Michon, Alexis; Osman, Roman; Orozco, Modesto; Perez, Alberto; Singh, Tanya; Spackova, Nada; Sponer, Jiri

2010-01-01

It is well recognized that base sequence exerts a significant influence on the properties of DNA and plays a significant role in protein–DNA interactions vital for cellular processes. Understanding and predicting base sequence effects requires an extensive structural and dynamic dataset which is currently unavailable from experiment. A consortium of laboratories was consequently formed to obtain this information using molecular simulations. This article describes results providing information not only on all 10 unique base pair steps, but also on all possible nearest-neighbor effects on these steps. These results are derived from simulations of 50–100 ns on 39 different DNA oligomers in explicit solvent and using a physiological salt concentration. We demonstrate that the simulations are converged in terms of helical and backbone parameters. The results show that nearest-neighbor effects on base pair steps are very significant, implying that dinucleotide models are insufficient for predicting sequence-dependent behavior. Flanking base sequences can notably lead to base pair step parameters in dynamic equilibrium between two conformational sub-states. Although this study only provides limited data on next-nearest-neighbor effects, we suggest that such effects should be analyzed before attempting to predict the sequence-dependent behavior of DNA. PMID:19850719

Skip residues modulate the structural properties of the myosin rod and guide thick filament assembly

DOE PAGES

Taylor, Keenan C.; Buvoli, Massimo; Korkmaz, Elif Nihal; ...

2015-07-06

The rod of sarcomeric myosins directs thick filament assembly and is characterized by the insertion of four skip residues that introduce discontinuities in the coiled-coil heptad repeats. We report in this paper that the regions surrounding the first three skip residues share high structural similarity despite their low sequence homology. Near each of these skip residues, the coiled-coil transitions to a nonclose-packed structure inducing local relaxation of the superhelical pitch. Moreover, molecular dynamics suggest that these distorted regions can assume different conformationally stable states. In contrast, the last skip residue region constitutes a true molecular hinge, providing C-terminal rod flexibility.more » Assembly of myosin with mutated skip residues in cardiomyocytes shows that the functional importance of each skip residue is associated with rod position and reveals the unique role of the molecular hinge in promoting myosin antiparallel packing. By defining the biophysical properties of the rod, the structures and molecular dynamic calculations presented here provide insight into thick filament formation, and highlight the structural differences occurring between the coiled-coils of myosin and the stereotypical tropomyosin. Finally, in addition to extending our knowledge into the conformational and biological properties of coiled-coil discontinuities, the molecular characterization of the four myosin skip residues also provides a guide to modeling the effects of rod mutations causing cardiac and skeletal myopathies.« less

Fluorescence-tunable Ag-DNA biosensor with tailored cytotoxicity for live-cell applications

NASA Astrophysics Data System (ADS)

Bossert, Nelli; de Bruin, Donny; Götz, Maria; Bouwmeester, Dirk; Heinrich, Doris

2016-11-01

DNA-stabilized silver clusters (Ag-DNA) show excellent promise as a multi-functional nanoagent for molecular investigations in living cells. The unique properties of these fluorescent nanomaterials allow for intracellular optical sensors with tunable cytotoxicity based on simple modifications of the DNA sequences. Three Ag-DNA nanoagent designs are investigated, exhibiting optical responses to the intracellular environments and sensing-capability of ions, functional inside living cells. Their sequence-dependent fluorescence responses inside living cells include (1) a strong splitting of the fluorescence peak for a DNA hairpin construct, (2) an excitation and emission shift of up to 120 nm for a single-stranded DNA construct, and (3) a sequence robust in fluorescence properties. Additionally, the cytotoxicity of these Ag-DNA constructs is tunable, ranging from highly cytotoxic to biocompatible Ag-DNA, independent of their optical sensing capability. Thus, Ag-DNA represents a versatile live-cell nanoagent addressable towards anti-cancer, patient-specific and anti-bacterial applications.

Rapid comparison of protein binding site surfaces with Property Encoded Shape Distributions (PESD)

PubMed Central

Das, Sourav; Kokardekar, Arshad

2009-01-01

Patterns in shape and property distributions on the surface of binding sites are often conserved across functional proteins without significant conservation of the underlying amino-acid residues. To explore similarities of these sites from the viewpoint of a ligand, a sequence and fold-independent method was created to rapidly and accurately compare binding sites of proteins represented by property-mapped triangulated Gauss-Connolly surfaces. Within this paradigm, signatures for each binding site surface are produced by calculating their property-encoded shape distributions (PESD), a measure of the probability that a particular property will be at a specific distance to another on the molecular surface. Similarity between the signatures can then be treated as a measure of similarity between binding sites. As postulated, the PESD method rapidly detected high levels of similarity in binding site surface characteristics even in cases where there was very low similarity at the sequence level. In a screening experiment involving each member of the PDBBind 2005 dataset as a query against the rest of the set, PESD was able to retrieve a binding site with identical E.C. (Enzyme Commission) numbers as the top match in 79.5% of cases. The ability of the method in detecting similarity in binding sites with low sequence conservations were compared with state-of-the-art binding site comparison methods. PMID:19919089

Noncanonical structures and their thermodynamics of DNA and RNA under molecular crowding: beyond the Watson-Crick double helix.

PubMed

Sugimoto, Naoki

2014-01-01

How does molecular crowding affect the stability of nucleic acid structures inside cells? Water is the major solvent component in living cells, and the properties of water in the highly crowded media inside cells differ from that in buffered solution. As it is difficult to measure the thermodynamic behavior of nucleic acids in cells directly and quantitatively, we recently developed a cell-mimicking system using cosolutes as crowding reagents. The influences of molecular crowding on the structures and thermodynamics of various nucleic acid sequences have been reported. In this chapter, we discuss how the structures and thermodynamic properties of nucleic acids differ under various conditions such as highly crowded environments, compartment environments, and in the presence of ionic liquids, and the major determinants of the crowding effects on nucleic acids are discussed. The effects of molecular crowding on the activities of ribozymes and riboswitches on noncanonical structures of DNA- and RNA-like quadruplexes that play important roles in transcription and translation are also described. © 2014 Elsevier Inc. All rights reserved.

Identification of a new Apscaviroid from Japanese persimmon.

PubMed

Nakaune, Ryoji; Nakano, Masaaki

2008-01-01

Three viroid-like sequences were detected from Japanese persimmon (Diospyrus kaki Thunb.) by RT-PCR using primers specific for members of the genus Apscaviroid. Based on the sequences, we determined the complete genomic sequences. Two had 92.1-94.3% sequence identity with citrus viroid OS (CVd-OS) and 91.4-96.3% identity with apple fruit crinkle viroid (AFCVd), respectively. Another one, tentatively named persimmon viroid (PVd), had 396 nucleotides and less than 70% sequence identity with known viroids. The secondary structure of PVd is proposed to be rod-like with extensive base pairing and contains the terminal conserved region and the central conserved region characteristic of the genus Apscaviroid. Moreover, we confirmed that the viroids, including PVd, are graft transmissible from persimmon to persimmon and that persimmon is a natural host of these viroids. According to its molecular and biological properties, PVd should be considered a member of a new species in the genus Apscaviroid.

Impact of commercial precooking of common bean (Phaseolus vulgaris) on the generation of peptides, after pepsin-pancreatin hydrolysis, capable to inhibit dipeptidyl peptidase-IV.

PubMed

Mojica, Luis; Chen, Karen; de Mejía, Elvira González

2015-01-01

The objective of this research was to determine the bioactive properties of the released peptides from commercially available precook common beans (Phaseolus vulgaris). Bioactive properties and peptide profiles were evaluated in protein hydrolysates of raw and commercially precooked common beans. Five varieties (Black, Pinto, Red, Navy, and Great Northern) were selected for protein extraction, protein and peptide molecular mass profiles, and peptide sequences. Potential bioactivities of hydrolysates, including antioxidant capacity and inhibition of α-amylase, α-glucosidase, dipeptidyl peptidase-IV (DPP-IV), and angiotensin converting enzyme I (ACE) were analyzed after digestion with pepsin/pancreatin. Hydrolysates from Navy beans were the most potent inhibitors of DPP-IV with no statistical differences between precooked and raw (IC50 = 0.093 and 0.095 mg protein/mL, respectively). α-Amylase inhibition was higher for raw Red, Navy and Great Northern beans (36%, 31%, 27% relative to acarbose (rel ac)/mg protein, respectively). α-Glucosidase inhibition among all bean hydrolysates did not show significant differences; however, inhibition values were above 40% rel ac/mg protein. IC50 values for ACE were not significantly different among all bean hydrolysates (range 0.20 to 0.34 mg protein/mL), except for Red bean that presented higher IC50 values. Peptide molecular mass profile ranged from 500 to 3000 Da. A total of 11 and 17 biologically active peptide sequences were identified in raw and precooked beans, respectively. Peptide sequences YAGGS and YAAGS from raw Great Northern and precooked Pinto showed similar amino acid sequences and same potential ACE inhibition activity. Processing did not affect the bioactive properties of released peptides from precooked beans. Commercially precooked beans could contribute to the intake of bioactive peptides and promote health. © 2014 Institute of Food Technologists®

Semi-Interpenetrating polymer network hydrogels based on aspen hemicellulose and chitosan: Effect of crosslinking sequence on hydrogel properties

Treesearch

Muzaffer Ahmet Karaaslan; Mandla A. Tshabalala; Gisela Buschle-Diller

2012-01-01

Semi-interpenetrating network hydrogel films were prepared using hemicellulose and chemically crosslinked chitosan. Hemicellulose was extracted from aspen by using a novel alkaline treatment and characterized by HPSEC, and consisted of a mixture of high and low molecular weight polymeric fractions. HPLC analysis of the acid hydrolysate of the hemicellulose showed that...

Rapid diversification of the cotton genus (Gossypium: Malvaceae) revealed by analysis of sixteen nuclear and chloroplast genes.

Treesearch

Richard C. Cronn; Randall L. Small; Tamara Hanselkorn; Jonathan F. Wendel

2002-01-01

Previous molecular phylogenetic studies have failed to resolve the branching order among the major cotton (Gossypium) lineages, and it has been unclear whether this reflects actual history (rapid radiation) or sampling properties of the genes evaluated. In this paper, we reconsider the phylogenetic relationships of diploid cotton genome groups using DNA sequences from...

New perspectives on bacterial ferredoxin evolution

NASA Technical Reports Server (NTRS)

George, D. G.; Hunt, L. T.; Yeh, L.-S. L.; Barker, W. C.

1985-01-01

Ferredoxins are low-molecular-weight, nonheme, iron proteins which function as electron carriers in a wide variety of electron transport chains. Howard et al. (1983) have suggested that the amino end of Azotobacter vinelandii ferredoxin shows a greater similarity to the carboxyl end of ferredoxin from Chromatium vinosum and that their half-chain sequences are homologous when the half-chains of either species are considered in inverse order. Examination of this proposition has made it necessary to reevaluate previous conclusions concerning the evolution of bacterial ferredoxin. Attention is given to the properties of the bacterial ferredoxin sequences, and the evolution of the bacterial ferredoxins.

Variability and molecular typing of the woody-tree infecting prunus necrotic ringspot ilarvirus.

PubMed

Vasková, D; Petrzik, K; Karesová, R

2000-01-01

The 3'-part of the movement protein gene, the intergenic region and the complete coat protein gene of sixteen isolates of Prunus necrotic ringspot virus (PNRSV) from five different host species from the Czech Republic were sequenced in order to search for the bases of extensive variability of viroses caused by this pathogen. According to phylogenetic analyses all the 46 isolates sequenced to date split into three main groups, which correlated to a certain extend with their geographic origin. Modelled serological properties showed that all the new isolates belong to one serotype.

The Genomes On Line Database (GOLD) v.2: a monitor of genome projects worldwide

PubMed Central

Liolios, Konstantinos; Tavernarakis, Nektarios; Hugenholtz, Philip; Kyrpides, Nikos C.

2006-01-01

The Genomes On Line Database (GOLD) is a web resource for comprehensive access to information regarding complete and ongoing genome sequencing projects worldwide. The database currently incorporates information on over 1500 sequencing projects, of which 294 have been completed and the data deposited in the public databases. GOLD v.2 has been expanded to provide information related to organism properties such as phenotype, ecotype and disease. Furthermore, project relevance and availability information is now included. GOLD is available at . It is also mirrored at the Institute of Molecular Biology and Biotechnology, Crete, Greece at PMID:16381880

Sequence comparison in the crossover region of an oncogenic avian retrovirus recombinant and its nononcogenic parent: Genetic regions that control growth rate and oncogenic potential

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

Tsichlis, P.N.; Donehower, L.; Hager, G.

1982-11-01

NTRE is an avian retrovirus recombinant of the endogeneous nononcogenic Rous-associated virus-0 (RAV-0) and the oncogenic, exogeneous, transformation-defective (td) Prague strain of Rous sarcoma virus B (td-PrRSV-B). Oligonucleotide mapping had shown that the recombinant virus is indistinguishable from its RAV-0 parent except for the 3'-end sequences, which were derived from td-PrRSV-B. However, the virus exhibits properties which are typical of an exogenous virus: it grows to high titers in tissue culture, and it is oncogenic in vivo. To accurately define the genetic region responsible for these properties, the authors determined the nucleotide sequences of the recombinant and its RAV-0 parentmore » by using molecular clones of their DNA. These were compared with sequences already available for PrRSV-C, a virus closely related to the exogenous parent td-PrRSV-B. The results suggested that the crossover event which generated NTRE 7 took place in a region -501 to -401 nucleotides from the 3' end of the td-PrRSV parental genome and that sequences to the right of the recombination region were responsible for its growth properties and oncogenic potential. Since the exogenous-virus-specific sequences are expected to be missing from transformation-defective mutants of the Schmidt-Ruppin strain of RSV, which, like other exogeneous viruses, grow to high tiers in tissue culture and are oncogenic in vivo, the authors concluded that the growth properties and oncogenic potential of the exogeneous viruses are determined by sequences in the U3 region of the long terminal repeat. However, the authors propose that the exogeneous-virus-specific region may play a role in determining the oncogenic spectrum of a given oncogenic virus.« less

Physicochemical properties of the modeled structure of astacin metalloprotease moulting enzyme NAS-36 and mapping the druggable allosteric space of Heamonchus contortus, Brugia malayi and Ceanorhabditis elegans via molecular dynamics simulation.

PubMed

Sharma, Om Prakash; Agrawal, Sonali; Kumar, M Suresh

2013-12-01

Nematodes represent the second largest phylum in the animal kingdom. It is the most abundant species (500,000) in the planet. It causes chronic, debilitating infections worldwide such as ascariasis, trichuriasis, hookworm, enterobiasis, strongyloidiasis, filariasis and trichinosis, among others. Molecular modeling tools can play an important role in the identification and structural investigation of molecular targets that can act as a vital candidate against filariasis. In this study, sequence analysis of NAS-36 from H. contortus (Heamonchus contortus), B. malayi (Brugia malayi) and C. elegans (Ceanorhabditis elegans) has been performed, in order to identify the conserved residues. Tertiary structure was developed for an insight into the molecular structure of the enzyme. Molecular Dynamics Simulation (MDS) studies have been carried out to analyze the stability and the physical properties of the proposed enzyme models in the H. contortus, B. malayi and C. elegans. Moreover, the drug binding sites have been mapped for inhibiting the function of NAS-36 enzyme. The molecular identity of this protease could eventually demonstrate how ex-sheathment is regulated, as well as provide a potential target of anthelmintics for the prevention of nematode infections.

Effect of Terminal Modification on the Molecular Assembly and Mechanical Properties of Protein-Based Block Copolymers.

PubMed

Jacobsen, Matthew M; Tokareva, Olena S; Ebrahimi, Davoud; Huang, Wenwen; Ling, Shengjie; Dinjaski, Nina; Li, David; Simon, Marc; Staii, Cristian; Buehler, Markus J; Kaplan, David L; Wong, Joyce Y

2017-09-01

Accurate prediction and validation of the assembly of bioinspired peptide sequences into fibers with defined mechanical characteristics would aid significantly in designing and creating materials with desired properties. This process may also be utilized to provide insight into how the molecular architecture of many natural protein fibers is assembled. In this work, computational modeling and experimentation are used in tandem to determine how peptide terminal modification affects a fiber-forming core domain. Modeling shows that increased terminal molecular weight and hydrophilicity improve peptide chain alignment under shearing conditions and promote consolidation of semicrystalline domains. Mechanical analysis shows acute improvements to strength and elasticity, but significantly reduced extensibility and overall toughness. These results highlight an important entropic function that terminal domains of fiber-forming peptides exhibit as chain alignment promoters, which ultimately has notable consequences on the mechanical behavior of the final fiber products. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Bioinformatics and molecular modeling in glycobiology

PubMed Central

Schloissnig, Siegfried

2010-01-01

The field of glycobiology is concerned with the study of the structure, properties, and biological functions of the family of biomolecules called carbohydrates. Bioinformatics for glycobiology is a particularly challenging field, because carbohydrates exhibit a high structural diversity and their chains are often branched. Significant improvements in experimental analytical methods over recent years have led to a tremendous increase in the amount of carbohydrate structure data generated. Consequently, the availability of databases and tools to store, retrieve and analyze these data in an efficient way is of fundamental importance to progress in glycobiology. In this review, the various graphical representations and sequence formats of carbohydrates are introduced, and an overview of newly developed databases, the latest developments in sequence alignment and data mining, and tools to support experimental glycan analysis are presented. Finally, the field of structural glycoinformatics and molecular modeling of carbohydrates, glycoproteins, and protein–carbohydrate interaction are reviewed. PMID:20364395

Lamination and mixing in laminar flows driven by Lorentz body forces

NASA Astrophysics Data System (ADS)

Rossi, L.; Doorly, D.; Kustrin, D.

2012-01-01

We present a new approach to the design of mixers. This approach relies on a sequence of tailored flows coupled with a new procedure to quantify the local degree of striation, called lamination. Lamination translates to the distance over which the molecular diffusion needs to act to finalise mixing. A novel in situ mixing is achieved by the tailored sequence of flows. This sequence is shown with the property that material lines and lamination grow exponentially, according to processes akin to the well-known baker's map. The degree of mixing (stirring coefficient) likewise shows exponential growth before the saturation of the stirring rate. Such saturation happens when the typical striations' thickness is smaller than the diffusion's length scale. Moreover, without molecular diffusion, the predicted striations' thickness would be smaller than the size of an atom of hydrogen within 40 flow turnover times. In fact, we conclude that about 3 minutes, i.e. 15 turnover times, are sufficient to mix species with very low diffusivities, e.g. suspensions of virus, bacteria, human cells, and DNA.

Sequence of the fhuE outer-membrane receptor gene of Escherichia coli K12 and properties of mutants.

PubMed

Sauer, M; Hantke, K; Braun, V

1990-03-01

The fhuE gene of Escherichia coli codes for an outer-membrane receptor protein required for the uptake of iron(III) via coprogen, ferrioxamine B and rhodotorulic acid. The amino acid sequence, deduced from the nucleotide sequence, consisted of 729 residues. The mature form, composed of 693 residues, has a calculated molecular weight of 77,453, which agrees with the molecular weight of 76,000 determined by polyacrylamide gel electrophoresis. The FhuE protein contains four regions of homology with other TonB-dependent receptors. A valine to proline exchange in the 'TonB box' abolished transport activity. Phenotypic revertants with substitutions of arginine, glutamine, or leucine at the valine position exhibited increasing iron-coprogen transport rates. Point mutations resulting in the replacement of glycine (127) in the second homology region with either alanine, aspartate, valine, asparagine or histidine exhibited decreased transport rates (listed in descending order). A truncated FhuE protein lacking 24 amino acids at the C-terminal end was exported to the periplasm but failed to be inserted into the outer membrane.

Molecular beacon sequence design algorithm.

PubMed

Monroe, W Todd; Haselton, Frederick R

2003-01-01

A method based on Web-based tools is presented to design optimally functioning molecular beacons. Molecular beacons, fluorogenic hybridization probes, are a powerful tool for the rapid and specific detection of a particular nucleic acid sequence. However, their synthesis costs can be considerable. Since molecular beacon performance is based on its sequence, it is imperative to rationally design an optimal sequence before synthesis. The algorithm presented here uses simple Microsoft Excel formulas and macros to rank candidate sequences. This analysis is carried out using mfold structural predictions along with other free Web-based tools. For smaller laboratories where molecular beacons are not the focus of research, the public domain algorithm described here may be usefully employed to aid in molecular beacon design.

Structural study of Bombyx mori silk fibroin during processing for regeneration

NASA Astrophysics Data System (ADS)

Ha, Sung-Won

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

Intraductal papillary-mucinous neoplasia of the pancreas: Histopathology and molecular biology.

PubMed

Verbeke, Caroline S

2010-10-27

Intraductal papillary-mucinous neoplasm (IPMN) of the pancreas is a clinically and morphologically distinctive precursor lesion of pancreatic cancer, characterized by gradual progression through a sequence of neoplastic changes. Based on the nature of the constituting neoplastic epithelium, degree of dysplasia and location within the pancreatic duct system, IPMNs are divided in several types which differ in their biological properties and clinical outcome. Molecular analysis and recent animal studies suggest that IPMNs develop in the context of a field-defect and reveal their possible relationship with other neoplastic precursor lesions of pancreatic cancer.

Genetic, genomic, and molecular tools for studying the protoploid yeast, L. waltii.

PubMed

Di Rienzi, Sara C; Lindstrom, Kimberly C; Lancaster, Ragina; Rolczynski, Lisa; Raghuraman, M K; Brewer, Bonita J

2011-02-01

Sequencing of the yeast Kluyveromyces waltii (recently renamed Lachancea waltii) provided evidence of a whole genome duplication event in the lineage leading to the well-studied Saccharomyces cerevisiae. While comparative genomic analyses of these yeasts have proven to be extremely instructive in modeling the loss or maintenance of gene duplicates, experimental tests of the ramifications following such genome alterations remain difficult. To transform L. waltii from an organism of the computational comparative genomic literature into an organism of the functional comparative genomic literature, we have developed genetic, molecular and genomic tools for working with L. waltii. In particular, we have characterized basic properties of L. waltii (growth, ploidy, molecular karyotype, mating type and the sexual cycle), developed transformation, cell cycle arrest and synchronization protocols, and have created centromeric and non-centromeric vectors as well as a genome browser for L. waltii. We hope that these tools will be used by the community to follow up on the ideas generated by sequence data and lead to a greater understanding of eukaryotic biology and genome evolution. 2010 John Wiley & Sons, Ltd.

Genetic, genomic, and molecular tools for studying the protoploid yeast, L. waltii

PubMed Central

Di Rienzi, Sara C.; Lindstrom, Kimberly C.; Lancaster, Ragina; Rolczynski, Lisa; Raghuraman, M. K.; Brewer, Bonita J.

2011-01-01

Sequencing of the yeast Kluyveromyces waltii (recently renamed Lachancea waltii) provided evidence of a whole genome duplication event in the lineage leading to the well-studied Saccharomyces cerevisiae. While comparative genomic analyses of these yeasts have proven to be extremely instructive in modeling the loss or maintenance of gene duplicates, experimental tests of the ramifications following such genome alterations remain difficult. To transform L. waltii from an organism of the computational comparative genomic literature into an organism of the functional comparative genomic literature, we have developed genetic, molecular and genomic tools for working with L. waltii. In particular, we have characterized basic properties of L. waltii (growth, ploidy, molecular karyotype, mating type and the sexual cycle), developed transformation, cell cycle arrest and synchronization protocols, and have created centromeric and non-centromeric vectors as well as a genome browser for L. waltii. We hope that these tools will be used by the community to follow up on the ideas generated by sequence data and lead to a greater understanding of eukaryotic biology and genome evolution. PMID:21246627

Sequence-Dependent Structure/Function Relationships of Catalytic Peptide-Enabled Gold Nanoparticles Generated under Ambient Synthetic Conditions

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

Bedford, Nicholas M.; Hughes, Zak E.; Tang, Zhenghua

Peptide-enabled nanoparticle (NP) synthesis routes can create and/or assemble functional nanomaterials under environmentally friendly conditions, with properties dictated by complex interactions at the biotic/abiotic interface. Manipulation of this interface through sequence modification can provide the capability for material properties to be tailored to create enhanced materials for energy, catalysis, and sensing applications. Fully realizing the potential of these materials requires a comprehensive understanding of sequence-dependent structure/function relationships that is presently lacking. In this work, the atomic-scale structures of a series of peptide-capped Au NPs are determined using a combination of atomic pair distribution function analysis of high-energy X-ray diffraction datamore » and advanced molecular dynamics (MD) simulations. The Au NPs produced with different peptide sequences exhibit varying degrees of catalytic activity for the exemplar reaction 4-nitrophenol reduction. The experimentally derived atomic-scale NP configurations reveal sequence-dependent differences in structural order at the NP surface. Replica exchange with solute-tempering MD simulations are then used to predict the morphology of the peptide overlayer on these Au NPs and identify factors determining the structure/catalytic properties relationship. We show that the amount of exposed Au surface, the underlying surface structural disorder, and the interaction strength of the peptide with the Au surface all influence catalytic performance. A simplified computational prediction of catalytic performance is developed that can potentially serve as a screening tool for future studies. Our approach provides a platform for broadening the analysis of catalytic peptide-enabled metallic NP systems, potentially allowing for the development of rational design rules for property enhancement.« less

Molecular evolution of FtsZ protein sequences encoded within the genomes of archaea, bacteria, and eukaryota.

PubMed

Vaughan, Sue; Wickstead, Bill; Gull, Keith; Addinall, Stephen G

2004-01-01

The FtsZ protein is a polymer-forming GTPase which drives bacterial cell division and is structurally and functionally related to eukaryotic tubulins. We have searched for FtsZ-related sequences in all freely accessible databases, then used strict criteria based on the tertiary structure of FtsZ and its well-characterized in vitro and in vivo properties to determine which sequences represent genuine homologues of FtsZ. We have identified 225 full-length FtsZ homologues, which we have used to document, phylum by phylum, the primary sequence characteristics of FtsZ homologues from the Bacteria, Archaea, and Eukaryota. We provide evidence for at least five independent ftsZ gene-duplication events in the bacterial kingdom and suggest the existence of three ancestoral euryarchaeal FtsZ paralogues. In addition, we identify "FtsZ-like" sequences from Bacteria and Archaea that, while showing significant sequence similarity to FtsZs, are unlikely to bind and hydrolyze GTP.

Unraveling the Molecular Requirements for Macroscopic Silk Supercontraction.

PubMed

Giesa, Tristan; Schuetz, Roman; Fratzl, Peter; Buehler, Markus J; Masic, Admir

2017-10-24

Spider dragline silk is a protein material that has evolved over millions of years to achieve finely tuned mechanical properties. A less known feature of some dragline silk fibers is that they shrink along the main axis by up to 50% when exposed to high humidity, a phenomenon called supercontraction. This contrasts the typical behavior of many other materials that swell when exposed to humidity. Molecular level details and mechanisms of the supercontraction effect are heavily debated. Here we report a molecular dynamics analysis of supercontraction in Nephila clavipes silk combined with in situ mechanical testing and Raman spectroscopy linking the reorganization of the nanostructure to the polar and charged amino acids in the sequence. We further show in our in silico approach that point mutations of these groups not only suppress the supercontraction effect, but even reverse it, while maintaining the exceptional mechanical properties of the silk material. This work has imminent impact on the design of biomimetic equivalents and recombinant silks for which supercontraction may or may not be a desirable feature. The approach applied is appropriate to explore the effect of point mutations on the overall physical properties of protein based materials.

Bioactive peptides from selected latin american food crops - A nutraceutical and molecular approach.

PubMed

Orona-Tamayo, Domancar; Valverde, María Elena; Paredes-López, Octavio

2018-02-01

This review reported an updated survey on the molecular functional properties of bioactive peptides derived from different Latin American ancient grains such as Maize, common Bean, Amaranth, Quinoa and Chia seeds. Seed storage proteins ecrypt in their sequences diverse peptides associated with a wide range of beneficial effects on the human health and the most studied are antihypertensive, anti-cholesterolemic, antioxidant, anti-inflammatory, anticancer, antimicrobial and immunomodulatory properties. Additionally, in the last decades molecular properties have been also used for their characterization to understand their activities and it makes them highly attractive to be incorporated into food formulations and to complement or replace some conventional cereal grains. Due to the nutraceutical effects, today, these seeds are one of the main gastronomic trends of consumption worldwide due to their nutritional benefits and are part of the shopping lists of many people, among them vegetarians, vegans, celiacs or lovers of raw food. These seeds are a legacy of pre-Columbian civilizations reason why in our time they are considered as "Superfoods of the Gods", "The pre-hispanic superfoods of the future" and "The new golden seeds of the XXI century".

25 years and still going strong: 2'-O-(pyren-1-yl)methylribonucleotides - versatile building blocks for applications in molecular biology, diagnostics and materials science.

PubMed

Hrdlicka, Patrick J; Karmakar, Saswata

2017-11-29

Oligonucleotides (ONs) modified with 2'-O-(pyren-1-yl)methylribonucleotides have been explored for a range of applications in molecular biology, nucleic acid diagnostics, and materials science for more than 25 years. The first part of this review provides an overview of synthetic strategies toward 2'-O-(pyren-1-yl)methylribonucleotides and is followed by a summary of biophysical properties of nucleic acid duplexes modified with these building blocks. Insights from structural studies are then presented to rationalize the reported properties. In the second part, applications of ONs modified with 2'-O-(pyren-1-yl)methyl-RNA monomers are reviewed, which include detection of RNA targets, discrimination of single nucleotide polymorphisms, formation of self-assembled pyrene arrays on nucleic acid scaffolds, the study of charge transfer phenomena in nucleic acid duplexes, and sequence-unrestricted recognition of double-stranded DNA. The predictable binding mode of the pyrene moiety, coupled with the microenvironment-dependent properties and synthetic feasibility, render 2'-O-(pyren-1-yl)methyl-RNA monomers as a promising class of pyrene-functionalized nucleotide building blocks for new applications in molecular biology, nucleic acid diagnostics, and materials science.

Nucleotide Sequence and Genetic Structure of a Novel Carbaryl Hydrolase Gene (cehA) from Rhizobium sp. Strain AC100

PubMed Central

Hashimoto, Masayuki; Fukui, Mitsuru; Hayano, Kouichi; Hayatsu, Masahito

2002-01-01

Rhizobium sp. strain AC100, which is capable of degrading carbaryl (1-naphthyl-N-methylcarbamate), was isolated from soil treated with carbaryl. This bacterium hydrolyzed carbaryl to 1-naphthol and methylamine. Carbaryl hydrolase from the strain was purified to homogeneity, and its N-terminal sequence, molecular mass (82 kDa), and enzymatic properties were determined. The purified enzyme hydrolyzed 1-naphthyl acetate and 4-nitrophenyl acetate indicating that the enzyme is an esterase. We then cloned the carbaryl hydrolase gene (cehA) from the plasmid DNA of the strain and determined the nucleotide sequence of the 10-kb region containing cehA. No homologous sequences were found by a database homology search using the nucleotide and deduced amino acid sequences of the cehA gene. Six open reading frames including the cehA gene were found in the 10-kb region, and sequencing analysis shows that the cehA gene is flanked by two copies of insertion sequence-like sequence, suggesting that it makes part of a composite transposon. PMID:11872471

Length and sequence dependence in the association of Huntingtin protein with lipid membranes

NASA Astrophysics Data System (ADS)

Jawahery, Sudi; Nagarajan, Anu; Matysiak, Silvina

2013-03-01

There is a fundamental gap in our understanding of how aggregates of mutant Huntingtin protein (htt) with overextended polyglutamine (polyQ) sequences gain the toxic properties that cause Huntington's disease (HD). Experimental studies have shown that the most important step associated with toxicity is the binding of mutant htt aggregates to lipid membranes. Studies have also shown that flanking amino acid sequences around the polyQ sequence directly affect interactions with the lipid bilayer, and that polyQ sequences of greater than 35 glutamine repeats in htt are a characteristic of HD. The key steps that determine how flanking sequences and polyQ length affect the structure of lipid bilayers remain unknown. In this study, we use atomistic molecular dynamics simulations to study the interactions between lipid membranes of varying compositions and polyQ peptides of varying lengths and flanking sequences. We find that overextended polyQ interactions do cause deformation in model membranes, and that the flanking sequences do play a role in intensifying this deformation by altering the shape of the affected regions.

A proposal to rationalize within-species plant virus nomenclature: benefits and implications of inaction.

PubMed

Jones, Roger A C; Kehoe, Monica A

2016-07-01

Current approaches used to name within-species, plant virus phylogenetic groups are often misleading and illogical. They involve names based on biological properties, sequence differences and geographical, country or place-association designations, or any combination of these. This type of nomenclature is becoming increasingly unsustainable as numbers of sequences of the same virus from new host species and different parts of the world increase. Moreover, this increase is accelerating as world trade and agriculture expand, and climate change progresses. Serious consequences for virus research and disease management might arise from incorrect assumptions made when current within-species phylogenetic group names incorrectly identify properties of group members. This could result in development of molecular tools that incorrectly target dangerous virus strains, potentially leading to unjustified impediments to international trade or failure to prevent such strains being introduced to countries, regions or continents formerly free of them. Dangerous strains might be missed or misdiagnosed by diagnostic laboratories and monitoring programs, and new cultivars with incorrect strain-specific resistances released. Incorrect deductions are possible during phylogenetic analysis of plant virus sequences and errors from strain misidentification during molecular and biological virus research activities. A nomenclature system for within-species plant virus phylogenetic group names is needed which avoids such problems. We suggest replacing all other naming approaches with Latinized numerals, restricting biologically based names only to biological strains and removing geographically based names altogether. Our recommendations have implications for biosecurity authorities, diagnostic laboratories, disease-management programs, plant breeders and researchers.

Intra-tumor heterogeneity in breast cancer has limited impact on transcriptomic-based molecular profiling.

PubMed

Karthik, Govindasamy-Muralidharan; Rantalainen, Mattias; Stålhammar, Gustav; Lövrot, John; Ullah, Ikram; Alkodsi, Amjad; Ma, Ran; Wedlund, Lena; Lindberg, Johan; Frisell, Jan; Bergh, Jonas; Hartman, Johan

2017-11-29

Transcriptomic profiling of breast tumors provides opportunity for subtyping and molecular-based patient stratification. In diagnostic applications the specimen profiled should be representative of the expression profile of the whole tumor and ideally capture properties of the most aggressive part of the tumor. However, breast cancers commonly exhibit intra-tumor heterogeneity at molecular, genomic and in phenotypic level, which can arise during tumor evolution. Currently it is not established to what extent a random sampling approach may influence molecular breast cancer diagnostics. In this study we applied RNA-sequencing to quantify gene expression in 43 pieces (2-5 pieces per tumor) from 12 breast tumors (Cohort 1). We determined molecular subtype and transcriptomic grade for all tumor pieces and analysed to what extent pieces originating from the same tumors are concordant or discordant with each other. Additionally, we validated our finding in an independent cohort consisting of 19 pieces (2-6 pieces per tumor) from 6 breast tumors (Cohort 2) profiled using microarray technique. Exome sequencing was also performed on this cohort, to investigate the extent of intra-tumor genomic heterogeneity versus the intra-tumor molecular subtype classifications. Molecular subtyping was consistent in 11 out of 12 tumors and transcriptomic grade assignments were consistent in 11 out of 12 tumors as well. Molecular subtype predictions revealed consistent subtypes in four out of six patients in this cohort 2. Interestingly, we observed extensive intra-tumor genomic heterogeneity in these tumor pieces but not in their molecular subtype classifications. Our results suggest that macroscopic intra-tumoral transcriptomic heterogeneity is limited and unlikely to have an impact on molecular diagnostics for most patients.

Prediction of the aggregation propensity of proteins from the primary sequence: aggregation properties of proteomes.

PubMed

Castillo, Virginia; Graña-Montes, Ricardo; Sabate, Raimon; Ventura, Salvador

2011-06-01

In the cell, protein folding into stable globular conformations is in competition with aggregation into non-functional and usually toxic structures, since the biophysical properties that promote folding also tend to favor intermolecular contacts, leading to the formation of β-sheet-enriched insoluble assemblies. The formation of protein deposits is linked to at least 20 different human disorders, ranging from dementia to diabetes. Furthermore, protein deposition inside cells represents a major obstacle for the biotechnological production of polypeptides. Importantly, the aggregation behavior of polypeptides appears to be strongly influenced by the intrinsic properties encoded in their sequences and specifically by the presence of selective short regions with high aggregation propensity. This allows computational methods to be used to analyze the aggregation properties of proteins without the previous requirement for structural information. Applications range from the identification of individual amyloidogenic regions in disease-linked polypeptides to the analysis of the aggregation properties of complete proteomes. Herein, we review these theoretical approaches and illustrate how they have become important and useful tools in understanding the molecular mechanisms underlying protein aggregation. Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Static electric polarizabilities and first hyperpolarizabilities of molecular ions RgH + (Rg = He, Ne, Ar, Kr, Xe): ab initio study

NASA Astrophysics Data System (ADS)

Cukras, Janusz; Antušek, Andrej; Holka, Filip; Sadlej, Joanna

2009-06-01

Extensive ab initio calculations of static electric properties of molecular ions of general formula RgH + (Rg = He, Ne, Ar, Kr, Xe) involving the finite field method and coupled cluster CCSD(T) approach have been done. The relativistic effects were taken into account by Douglas-Kroll-Hess approximation. The numerical stability and reliability of calculated values have been tested using the systematic sequence of Dunning's cc-pVXZ-DK and ANO-RCC-VQZP basis sets. The influence of ZPE and pure vibrational contribution has been discussed. The component αzz has increasing trend in RgH + while the relativistic effect on αzz leads to a small increase of this molecular parameter.

Adhesive barnacle peptides exhibit a steric-driven design rule to enhance adhesion between asymmetric surfaces.

PubMed

Raman, Sangeetha; Malms, Lukas; Utzig, Thomas; Shrestha, Buddha Ratna; Stock, Philipp; Krishnan, Shankar; Valtiner, Markus

2017-04-01

Barnacles exhibit superior underwater adhesion simply through sequencing of the 21 proteinogenic amino acids, without post processing or using special amino acids. Here, we measure and discuss the molecular interaction of two distinct and recurring short peptide sequences (Bp1 and Bp2) inspired from the surface binding 19kDa protein from the barnacle attachment interface. Using self-assembled monolayer (SAMs) of known physical and chemical properties on molecularly smooth gold substrates in 5mM NaCl at pH 7.3, (1) the adsorption mechanisms of the barnacle inspired peptides are explored using quartz crystal microbalance, and (2) adhesion mediating properties are measured using the surface force apparatus. The hydrophobic Bp1 peptide with a cysteine residue adsorbs irreversibly onto Au surfaces due to thiol bond formation, while on hydrophobic CH 3 SAM surface, the interactions are hydrophobic in nature. Interestingly, Bp2 that contains both hydrophobic and protonated amine units exhibits asymmetric bridging with an exceptionally high adhesion energy up to 100mJ/m 2 between mica and both gold and CH 3 SAM. Surprisingly on hydrophilic surfaces such as COOH- or OH-SAMs both peptides fail to show any interactions, implying the necessity of surface charge to promote bridging. Our results provide insights into the molecular aspects of manipulating and utilizing barnacle-mediated peptides to promote or inhibit underwater adhesion. Copyright © 2016 Elsevier B.V. All rights reserved.

Fast Homozygosity Mapping and Identification of a Zebrafish ENU-Induced Mutation by Whole-Genome Sequencing

PubMed Central

Voz, Marianne L.; Coppieters, Wouter; Manfroid, Isabelle; Baudhuin, Ariane; Von Berg, Virginie; Charlier, Carole; Meyer, Dirk; Driever, Wolfgang; Martial, Joseph A.; Peers, Bernard

2012-01-01

Forward genetics using zebrafish is a powerful tool for studying vertebrate development through large-scale mutagenesis. Nonetheless, the identification of the molecular lesion is still laborious and involves time-consuming genetic mapping. Here, we show that high-throughput sequencing of the whole zebrafish genome can directly locate the interval carrying the causative mutation and at the same time pinpoint the molecular lesion. The feasibility of this approach was validated by sequencing the m1045 mutant line that displays a severe hypoplasia of the exocrine pancreas. We generated 13 Gb of sequence, equivalent to an eightfold genomic coverage, from a pool of 50 mutant embryos obtained from a map-cross between the AB mutant carrier and the WIK polymorphic strain. The chromosomal region carrying the causal mutation was localized based on its unique property to display high levels of homozygosity among sequence reads as it derives exclusively from the initial AB mutated allele. We developed an algorithm identifying such a region by calculating a homozygosity score along all chromosomes. This highlighted an 8-Mb window on chromosome 5 with a score close to 1 in the m1045 mutants. The sequence analysis of all genes within this interval revealed a nonsense mutation in the snapc4 gene. Knockdown experiments confirmed the assertion that snapc4 is the gene whose mutation leads to exocrine pancreas hypoplasia. In conclusion, this study constitutes a proof-of-concept that whole-genome sequencing is a fast and effective alternative to the classical positional cloning strategies in zebrafish. PMID:22496837

Molecular determinants of the V3 loop of human immunodeficiency virus type 1 glycoprotein gp120 responsible for controlling cell tropism.

PubMed

Chavda, S C; Griffin, P; Han-Liu, Z; Keys, B; Vekony, M A; Cann, A J

1994-11-01

We and others have identified the major determinant of cell tropism in human immunodeficiency virus type 1 (HIV-1) as the V3 loop of glycoprotein gp120. We have conducted a detailed study of two molecularly cloned isolates of HIV-1, HIVJR-CSF and HIVNL4-3, that differ in their tropism for immortalized CD4+ cell lines, by constructing a series of site-directed mutations within the V3 loop of HIVJR-CSF based on the sequence of HIVNL4-3. The phenotypes of these mutants fall into two classes, those which are viable and those which are not. A spontaneous mutant with significantly altered growth properties was also recovered and found to have an additional single amino acid change in the V3 loop sequence. The carboxy-terminal beta-strand part of the V3 loop is the major determinant of cell tropism. However, the results presented here indicate that the functional role of the V3 loop sequences can only be interpreted properly in the context of the original gp120 backbone from which they were derived. These findings show that over-simplistic interpretation of sequence data derived from unknown mixtures of HIV variants in infected persons may be highly misleading.

Draft genome sequence of an inbred line of Chenopodium quinoa, an allotetraploid crop with great environmental adaptability and outstanding nutritional properties

PubMed Central

Yasui, Yasuo; Hirakawa, Hideki; Oikawa, Tetsuo; Toyoshima, Masami; Matsuzaki, Chiaki; Ueno, Mariko; Mizuno, Nobuyuki; Nagatoshi, Yukari; Imamura, Tomohiro; Miyago, Manami; Tanaka, Kojiro; Mise, Kazuyuki; Tanaka, Tsutomu; Mizukoshi, Hiroharu; Mori, Masashi; Fujita, Yasunari

2016-01-01

Chenopodium quinoa Willd. (quinoa) originated from the Andean region of South America, and is a pseudocereal crop of the Amaranthaceae family. Quinoa is emerging as an important crop with the potential to contribute to food security worldwide and is considered to be an optimal food source for astronauts, due to its outstanding nutritional profile and ability to tolerate stressful environments. Furthermore, plant pathologists use quinoa as a representative diagnostic host to identify virus species. However, molecular analysis of quinoa is limited by its genetic heterogeneity due to outcrossing and its genome complexity derived from allotetraploidy. To overcome these obstacles, we established the inbred and standard quinoa accession Kd that enables rigorous molecular analysis, and presented the draft genome sequence of Kd, using an optimized combination of high-throughput next generation sequencing on the Illumina Hiseq 2500 and PacBio RS II sequencers. The de novo genome assembly contained 25 k scaffolds consisting of 1 Gbp with N50 length of 86 kbp. Based on these data, we constructed the free-access Quinoa Genome DataBase (QGDB). Thus, these findings provide insights into the mechanisms underlying agronomically important traits of quinoa and the effect of allotetraploidy on genome evolution. PMID:27458999

Evolutionary Dynamics of the Gametologous CTNNB1 Gene on the Z and W Chromosomes of Snakes.

PubMed

Laopichienpong, Nararat; Muangmai, Narongrit; Chanhome, Lawan; Suntrarachun, Sunutcha; Twilprawat, Panupon; Peyachoknagul, Surin; Srikulnath, Kornsorn

2017-03-01

Snakes exhibit genotypic sex determination with female heterogamety (ZZ males and ZW females), and the state of sex chromosome differentiation also varies among lineages. To investigate the evolutionary history of homologous genes located in the nonrecombining region of differentiated sex chromosomes in snakes, partial sequences of the gametologous CTNNB1 gene were analyzed for 12 species belonging to henophid (Cylindrophiidae, Xenopeltidae, and Pythonidae) and caenophid snakes (Viperidae, Elapidae, and Colubridae). Nonsynonymous/synonymous substitution ratios (Ka/Ks) in coding sequences were low (Ka/Ks < 1) between CTNNB1Z and CTNNB1W, suggesting that these 2 genes may have similar functional properties. However, frequencies of intron sequence substitutions and insertion–deletions were higher in CTNNB1Z than CTNNB1W, suggesting that Z-linked sequences evolved faster than W-linked sequences. Molecular phylogeny based on both intron and exon sequences showed the presence of 2 major clades: 1) Z-linked sequences of Caenophidia and 2) W-linked sequences of Caenophidia clustered with Z-linked sequences of Henophidia, which suggests that the sequence divergence between CTNNB1Z and CTNNB1W in Caenophidia may have occurred by the cessation of recombination after the split from Henophidia.

Protein mechanics: from single molecules to functional biomaterials.

PubMed

Li, Hongbin; Cao, Yi

2010-10-19

Elastomeric proteins act as the essential functional units in a wide variety of biomechanical machinery and serve as the basic building blocks for biological materials that exhibit superb mechanical properties. These proteins provide the desired elasticity, mechanical strength, resilience, and toughness within these materials. Understanding the mechanical properties of elastomeric protein-based biomaterials is a multiscale problem spanning from the atomistic/molecular level to the macroscopic level. Uncovering the design principles of individual elastomeric building blocks is critical both for the scientific understanding of multiscale mechanics of biomaterials and for the rational engineering of novel biomaterials with desirable mechanical properties. The development of single-molecule force spectroscopy techniques has provided methods for characterizing mechanical properties of elastomeric proteins one molecule at a time. Single-molecule atomic force microscopy (AFM) is uniquely suited to this purpose. Molecular dynamic simulations, protein engineering techniques, and single-molecule AFM study have collectively revealed tremendous insights into the molecular design of single elastomeric proteins, which can guide the design and engineering of elastomeric proteins with tailored mechanical properties. Researchers are focusing experimental efforts toward engineering artificial elastomeric proteins with mechanical properties that mimic or even surpass those of natural elastomeric proteins. In this Account, we summarize our recent experimental efforts to engineer novel artificial elastomeric proteins and develop general and rational methodologies to tune the nanomechanical properties of elastomeric proteins at the single-molecule level. We focus on general design principles used for enhancing the mechanical stability of proteins. These principles include the development of metal-chelation-based general methodology, strategies to control the unfolding hierarchy of multidomain elastomeric proteins, and the design of novel elastomeric proteins that exhibit stimuli-responsive mechanical properties. Moving forward, we are now exploring the use of these artificial elastomeric proteins as building blocks of protein-based biomaterials. Ultimately, we would like to rationally tailor mechanical properties of elastomeric protein-based materials by programming the molecular sequence, and thus nanomechanical properties, of elastomeric proteins at the single-molecule level. This step would help bridge the gap between single protein mechanics and material biomechanics, revealing how the mechanical properties of individual elastomeric proteins are translated into the properties of macroscopic materials.

Molecular dynamics study of binding energies, mechanical properties, and detonation performances of bicyclo-HMX-based PBXs.

PubMed

Qiu, Ling; Xiao, Heming

2009-05-15

To investigate the effect of polymer binders on the monoexplosive, molecular dynamics simulations were performed to study the binding energies, mechanical properties, and detonation performances of the bicyclo-HMX-based polymer-bonded explosives (PBXs). The results show that the binding energies on different crystalline surfaces of bicyclo-HMX decrease in the order of (010)>(100)>(001). On each crystalline surface, binding properties of different polymers with the same chain segment are different from each other, while those of the polymers in the same content decrease in the sequence of PVDF>F(2311)>F(2314) approximately PCTFE. The mechanical properties of a dozen of model systems (elastic coefficients, various moduli, Cauchy pressure, and Poisson's ratio) have been obtained. It is found that mechanical properties are effectively improved by adding small amounts of fluorine polymers, and the overall effect of fluorine polymers on three crystalline surfaces of bicyclo-HMX changes in the order of (010)>(001) approximately (100). In comparison with the base explosive, detonation performances of the PBXs decrease slightly, but they are still superior to TNT. These suggestions may be useful for the formulation design of bicyclo-HMX-based PBXs.

Guiding principles for peptide nanotechnology through directed discovery.

PubMed

Lampel, A; Ulijn, R V; Tuttle, T

2018-05-21

Life's diverse molecular functions are largely based on only a small number of highly conserved building blocks - the twenty canonical amino acids. These building blocks are chemically simple, but when they are organized in three-dimensional structures of tremendous complexity, new properties emerge. This review explores recent efforts in the directed discovery of functional nanoscale systems and materials based on these same amino acids, but that are not guided by copying or editing biological systems. The review summarises insights obtained using three complementary approaches of searching the sequence space to explore sequence-structure relationships for assembly, reactivity and complexation, namely: (i) strategic editing of short peptide sequences; (ii) computational approaches to predicting and comparing assembly behaviours; (iii) dynamic peptide libraries that explore the free energy landscape. These approaches give rise to guiding principles on controlling order/disorder, complexation and reactivity by peptide sequence design.

Biocuration in the structure-function linkage database: the anatomy of a superfamily.

PubMed

Holliday, Gemma L; Brown, Shoshana D; Akiva, Eyal; Mischel, David; Hicks, Michael A; Morris, John H; Huang, Conrad C; Meng, Elaine C; Pegg, Scott C-H; Ferrin, Thomas E; Babbitt, Patricia C

2017-01-01

With ever-increasing amounts of sequence data available in both the primary literature and sequence repositories, there is a bottleneck in annotating molecular function to a sequence. This article describes the biocuration process and methods used in the structure-function linkage database (SFLD) to help address some of the challenges. We discuss how the hierarchy within the SFLD allows us to infer detailed functional properties for functionally diverse enzyme superfamilies in which all members are homologous, conserve an aspect of their chemical function and have associated conserved structural features that enable the chemistry. Also presented is the Enzyme Structure-Function Ontology (ESFO), which has been designed to capture the relationships between enzyme sequence, structure and function that underlie the SFLD and is used to guide the biocuration processes within the SFLD. http://sfld.rbvi.ucsf.edu/. © The Author 2017. Published by Oxford University Press.

Structural analysis as an alternative to identify and determine mode of action of antimicrobial peptides: proposition of a kinetic model based on molecular dynamics studies.

PubMed

Robles-Gomez, Edson Edinho; Flores-Villegas, Mirelle Citlali; Gonzalez-Manjarrez, Alicia; Soriano-Garcia, Manuel

2013-05-01

Antimicrobial peptides (AMPs) constitute an important alternative in the search for new treatments against pathogens. We analyzed the sequence variability in cytokine and chemokine proteins to investigate whether these molecules contain a sequence useful in the development of new AMPs. Cluster analysis allowed the identification of tracts, grouped in five categories showing structure and sequence homology. The structure and function relationship among these groups, was analyzed using physicochemical parameters such as length, sequence, charge, hydrophobicity and helicity, which allowed the selection of a candidate that could constitute an AMP. This peptide comprises the C-terminal alpha-helix of chemokines CXCL4/PF-457-70. Far-UV CD spectroscopy showed that this molecule adopts a random conformation in aqueous solution and the addition of 2, 2, 2 trifluoroethanol (TFE) is required to induce a helical secondary structure. The CXCL4/PF-457-70 peptide was found to have antimicrobial activity and very limited hemolytic activity. The mechanism of action was analyzed using model kinetics and molecular dynamics. The kinetic model led to a reasonable assumption about a rate constant and regulatory step on its mechanism of action. Using molecular dynamics simulations, the structural properties the CXCL4/PF-457-70 have been examined in a membrane environment. Our results show that this peptide has a strong preference for binding to the lipid head groups, consequently, increasing the surface density and decreasing the lateral mobility of the lipids alters its functionality.

Hepatitis A virus: host interactions, molecular epidemiology and evolution.

PubMed

Vaughan, Gilberto; Goncalves Rossi, Livia Maria; Forbi, Joseph C; de Paula, Vanessa S; Purdy, Michael A; Xia, Guoliang; Khudyakov, Yury E

2014-01-01

Infection with hepatitis A virus (HAV) is the commonest viral cause of liver disease and presents an important public health problem worldwide. Several unique HAV properties and molecular mechanisms of its interaction with host were recently discovered and should aid in clarifying the pathogenesis of hepatitis A. Genetic characterization of HAV strains have resulted in the identification of different genotypes and subtypes, which exhibit a characteristic worldwide distribution. Shifts in HAV endemicity occurring in different parts of the world, introduction of genetically diverse strains from geographically distant regions, genotype displacement observed in some countries and population expansion detected in the last decades of the 20th century using phylogenetic analysis are important factors contributing to the complex dynamics of HAV infections worldwide. Strong selection pressures, some of which, like usage of deoptimized codons, are unique to HAV, limit genetic variability of the virus. Analysis of subgenomic regions has been proven useful for outbreak investigations. However, sharing short sequences among epidemiologically unrelated strains indicates that specific identification of HAV strains for molecular surveillance can be achieved only using whole-genome sequences. Here, we present up-to-date information on the HAV molecular epidemiology and evolution, and highlight the most relevant features of the HAV-host interactions. Published by Elsevier B.V.

Molecular sled sequences are common in mammalian proteins.

PubMed

Xiong, Kan; Blainey, Paul C

2016-03-18

Recent work revealed a new class of molecular machines called molecular sleds, which are small basic molecules that bind and slide along DNA with the ability to carry cargo along DNA. Here, we performed biochemical and single-molecule flow stretching assays to investigate the basis of sliding activity in molecular sleds. In particular, we identified the functional core of pVIc, the first molecular sled characterized; peptide functional groups that control sliding activity; and propose a model for the sliding activity of molecular sleds. We also observed widespread DNA binding and sliding activity among basic polypeptide sequences that implicate mammalian nuclear localization sequences and many cell penetrating peptides as molecular sleds. These basic protein motifs exhibit weak but physiologically relevant sequence-nonspecific DNA affinity. Our findings indicate that many mammalian proteins contain molecular sled sequences and suggest the possibility that substantial undiscovered sliding activity exists among nuclear mammalian proteins. © The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.

Gas Content and Kinematics in Clumpy, Turbulent Star-forming Disks

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

White, Heidi A.; Abraham, Roberto G.; Fisher, David B.

2017-09-01

We present molecular gas-mass estimates for a sample of 13 local galaxies whose kinematic and star-forming properties closely resemble those observed in z ≈ 1.5 main-sequence galaxies. Plateau de Bure observations of the CO[1-0] emission line and Herschel Space Observatory observations of the dust emission both suggest molecular gas-mass fractions of ∼20%. Moreover, dust emission modeling finds T {sub dust} < 30 K, suggesting a cold dust distribution compared to their high infrared luminosity. The gas-mass estimates argue that z ∼ 0.1 DYNAMO galaxies not only share similar kinematic properties with high- z disks, but they are also similarly richmore » in molecular material. Pairing the gas-mass fractions with existing kinematics reveals a linear relationship between f {sub gas} and σ / v {sub c}, consistent with predictions from stability theory of a self-gravitating disk. It thus follows that high gas-velocity dispersions are a natural consequence of large gas fractions. We also find that the systems with the lowest t {sub dep} (∼0.5 Gyr) have the highest ratios of σ / v{sub c} and more pronounced clumps, even at the same high molecular gas fraction.« less

Isolation and molecular cloning of a fast-growing strain of human hepatitis A virus from its double-stranded replicative form.

PubMed Central

Venuti, A; Di Russo, C; del Grosso, N; Patti, A M; Ruggeri, F; De Stasio, P R; Martiniello, M G; Pagnotti, P; Degener, A M; Midulla, M

1985-01-01

A fast-growing strain of human hepatitis A virus was selected and characterized. The virus has the unusual property of developing a strong cytopathic effect in tissue culture in 7 to 10 days. Sequences of the viral genome were cloned into recombinant plasmids with the double-stranded replicative form as a template for the reverse transcription of cDNA. Restriction analysis and direct sequencing indicate that this strain is different from that described by Ticehurst et al. (Proc. Natl. Acad. Sci. USA 80:5885-5889, 1983) in the region that presumptively codes for the major capsid protein VP1, but both isolates have conserved large areas of homology in the untranslated 5'-terminal sequences of the genome. Images PMID:2997478

Generation of non-genomic oligonucleotide tag sequences for RNA template-specific PCR

PubMed Central

Pinto, Fernando Lopes; Svensson, Håkan; Lindblad, Peter

2006-01-01

Background In order to overcome genomic DNA contamination in transcriptional studies, reverse template-specific polymerase chain reaction, a modification of reverse transcriptase polymerase chain reaction, is used. The possibility of using tags whose sequences are not found in the genome further improves reverse specific polymerase chain reaction experiments. Given the absence of software available to produce genome suitable tags, a simple tool to fulfill such need was developed. Results The program was developed in Perl, with separate use of the basic local alignment search tool, making the tool platform independent (known to run on Windows XP and Linux). In order to test the performance of the generated tags, several molecular experiments were performed. The results show that Tagenerator is capable of generating tags with good priming properties, which will deliberately not result in PCR amplification of genomic DNA. Conclusion The program Tagenerator is capable of generating tag sequences that combine genome absence with good priming properties for RT-PCR based experiments, circumventing the effects of genomic DNA contamination in an RNA sample. PMID:16820068

Systematic analysis of snake neurotoxins' functional classification using a data warehousing approach.

PubMed

Siew, Joyce Phui Yee; Khan, Asif M; Tan, Paul T J; Koh, Judice L Y; Seah, Seng Hong; Koo, Chuay Yeng; Chai, Siaw Ching; Armugam, Arunmozhiarasi; Brusic, Vladimir; Jeyaseelan, Kandiah

2004-12-12

Sequence annotations, functional and structural data on snake venom neurotoxins (svNTXs) are scattered across multiple databases and literature sources. Sequence annotations and structural data are available in the public molecular databases, while functional data are almost exclusively available in the published articles. There is a need for a specialized svNTXs database that contains NTX entries, which are organized, well annotated and classified in a systematic manner. We have systematically analyzed svNTXs and classified them using structure-function groups based on their structural, functional and phylogenetic properties. Using conserved motifs in each phylogenetic group, we built an intelligent module for the prediction of structural and functional properties of unknown NTXs. We also developed an annotation tool to aid the functional prediction of newly identified NTXs as an additional resource for the venom research community. We created a searchable online database of NTX proteins sequences (http://research.i2r.a-star.edu.sg/Templar/DB/snake_neurotoxin). This database can also be found under Swiss-Prot Toxin Annotation Project website (http://www.expasy.org/sprot/).

Charge pattern matching as a ‘fuzzy’ mode of molecular recognition for the functional phase separations of intrinsically disordered proteins

NASA Astrophysics Data System (ADS)

Lin, Yi-Hsuan; Brady, Jacob P.; Forman-Kay, Julie D.; Chan, Hue Sun

2017-11-01

Biologically functional liquid-liquid phase separation of intrinsically disordered proteins (IDPs) is driven by interactions encoded by their amino acid sequences. Little is currently known about the molecular recognition mechanisms for distributing different IDP sequences into various cellular membraneless compartments. Pertinent physics was addressed recently by applying random-phase-approximation (RPA) polymer theory to electrostatics, which is a major energetic component governing IDP phase properties. RPA accounts for charge patterns and thus has advantages over Flory-Huggins (FH) and Overbeek-Voorn mean-field theories. To make progress toward deciphering the phase behaviors of multiple IDP sequences, the RPA formulation for one IDP species plus solvent is hereby extended to treat polyampholyte solutions containing two IDP species plus solvent. The new formulation generally allows for binary coexistence of two phases, each containing a different set of volume fractions ({φ }1,{φ }2) for the two different IDP sequences. The asymmetry between the two predicted coexisting phases with regard to their {φ }1/{φ }2 ratios for the two sequences increases with increasing mismatch between their charge patterns. This finding points to a multivalent, stochastic, ‘fuzzy’ mode of molecular recognition that helps populate various IDP sequences differentially into separate phase compartments. An intuitive illustration of this trend is provided by FH models, whereby a hypothetical case of ternary coexistence is also explored. Augmentations of the present RPA theory with a relative permittivity {ɛ }{{r}}(φ ) that depends on IDP volume fraction φ ={φ }1+{φ }2 lead to higher propensities to phase separate, in line with the case with one IDP species we studied previously. Notably, the cooperative, phase-separation-enhancing effects predicted by the prescriptions for {ɛ }{{r}}(φ ) we deem physically plausible are much more prominent than that entailed by common effective medium approximations based on Maxwell Garnett and Bruggeman mixing formulas. Ramifications of our findings on further theoretical development for IDP phase separation are discussed.

[Prospects of molecular breeding in medical plants].

PubMed

Ma, Xiao-Jun; Mo, Chang-Ming

2017-06-01

The molecular-assisted breeding, transgenic breeding and molecular designing breeding are three development directions of plant molecular breeding. Base on these three development directions, this paper summarizes developing status and new tendency of research field of genetic linkage mapping, QTL mapping, association mapping, molecular-assisted selections, pollen-mediated transformations, agrobacterium-mediated transformations, particle gun-mediated transformations, genome editing technologies, whole-genome sequencing, transcriptome sequencing, proteome sequencing and varietal molecular designing. The objective and existing problem of medical plant molecular breeding were discussed the prospect of these three molecular breeding technologies application on medical plant molecular breeding was outlooked. Copyright© by the Chinese Pharmaceutical Association.

An atomic scale study of surface termination and digital alloy growth in InGaAs/AlAsSb multi-quantum wells.

PubMed

Mauger, S J C; Bozkurt, M; Koenraad, P M; Zhao, Y; Folliot, H; Bertru, N

2016-07-20

An atomic scale study has been performed to understand the influence of the (As,Sb) shutter sequences during interface formation on the optical properties of InGaAs/AlAsSb quantum wells. Our cross-sectional scanning tunneling microscopy results show that the onset of the Sb profile is steep in the Sb-containing layers whereas an appreciable segregation of Sb in the subsequently grown Sb free layers is observed. The steep rise of the Sb profile is due to extra Sb that is supplied to the surface prior to the growth of the Sb-containing layers. No relation is found between the (As,Sb) termination conditions of the Sb-containing layers and the resulting Sb profiles in the capping layers. Correspondingly we see that the optical properties of these quantum wells are also nearly independent on the (As,Sb) shutter sequences at the interface. Digital alloy growth in comparison to conventional molecular beam epitaxy growth was also explored. X-ray results suggest that the structural properties of the quantum well structures grown by conventional molecular beam epitaxy techniques are slightly better than those formed by digital alloy growth. However photoluminescence studies indicate that the digital alloy samples give rise to a more intense and broader photoluminescence emission. Cross-sectional scanning tunneling microscopy measurements reveal that lateral composition modulations present in the digital alloys are responsible for the enhancement of the photoluminescence intensity and inhomogeneous broadening.

In silico selection of therapeutic antibodies for development: Viscosity, clearance, and chemical stability

PubMed Central

Sharma, Vikas K.; Patapoff, Thomas W.; Kabakoff, Bruce; Pai, Satyan; Hilario, Eric; Zhang, Boyan; Li, Charlene; Borisov, Oleg; Kelley, Robert F.; Chorny, Ilya; Zhou, Joe Z.; Dill, Ken A.; Swartz, Trevor E.

2014-01-01

For mAbs to be viable therapeutics, they must be formulated to have low viscosity, be chemically stable, and have normal in vivo clearance rates. We explored these properties by observing correlations of up to 60 different antibodies of the IgG1 isotype. Unexpectedly, we observe significant correlations with simple physical properties obtainable from antibody sequences and by molecular dynamics simulations of individual antibody molecules. mAbs viscosities increase strongly with hydrophobicity and charge dipole distribution and decrease with net charge. Fast clearance correlates with high hydrophobicities of certain complementarity determining regions and with high positive or high negative net charge. Chemical degradation from tryptophan oxidation correlates with the average solvent exposure time of tryptophan residues. Aspartic acid isomerization rates can be predicted from solvent exposure and flexibility as determined by molecular dynamics simulations. These studies should aid in more rapid screening and selection of mAb candidates during early discovery. PMID:25512516

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

NASA Astrophysics Data System (ADS)

Sethaphong, Latsavongsakda

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

[Analysis of structural characteristics of alpha-tubulins in plants with enhanced cold tolerance].

PubMed

Nyporko, A Iu; Demchuk, O N; Blium, Ia B

2003-01-01

The uniqueness of the point substitutions in the sequences of two alpha-tubulin isotypes from psychrophilic alga Chloromonas that can determine the increased cold tolerance of this alga was analyzed. The comparison of all known amino acid sequences of plant alpha-tubulins enabled to ascertain that only M268-->V replacement is unique and may have a significant influence on spatial structure of plant alpha-tubulins. Modeling of molecular surfaces of alpha-tubulins from Chloromonas, Chalmydomonas reinhardtii and goose grass Eleusine indica showed that insertion of the amino acid replacement M268-->V into the sequence of goose grace tubulin led to the likening of this protein surface to the surface of native alpha-tubulin from Chloromonas. Alteration of local hydrophobic properties of alpha-tubulin molecular surface in interdimeric contact zone as a result of the mentioned replacement was shown that may play important role in increasing the level of cold resistance of microtubules. The crucial role of amino acid residue in 268 position for forming the interdimeric contact surface of alpha-tubulin molecule was revealed. The assumption is made about the importance of replacements at this position for plant tolerance to abiotic factors of different nature (cold, herbicides).

Conformational gating of DNA conductance

PubMed Central

Artés, Juan Manuel; Li, Yuanhui; Qi, Jianqing; Anantram, M. P.; Hihath, Joshua

2015-01-01

DNA is a promising molecule for applications in molecular electronics because of its unique electronic and self-assembly properties. Here we report that the conductance of DNA duplexes increases by approximately one order of magnitude when its conformation is changed from the B-form to the A-form. This large conductance increase is fully reversible, and by controlling the chemical environment, the conductance can be repeatedly switched between the two values. The conductance of the two conformations displays weak length dependencies, as is expected for guanine-rich sequences, and can be fit with a coherence-corrected hopping model. These results are supported by ab initio electronic structure calculations that indicate that the highest occupied molecular orbital is more disperse in the A-form DNA case. These results demonstrate that DNA can behave as a promising molecular switch for molecular electronics applications and also provide additional insights into the huge dispersion of DNA conductance values found in the literature. PMID:26648400

Conformational gating of DNA conductance.

PubMed

Artés, Juan Manuel; Li, Yuanhui; Qi, Jianqing; Anantram, M P; Hihath, Joshua

2015-12-09

DNA is a promising molecule for applications in molecular electronics because of its unique electronic and self-assembly properties. Here we report that the conductance of DNA duplexes increases by approximately one order of magnitude when its conformation is changed from the B-form to the A-form. This large conductance increase is fully reversible, and by controlling the chemical environment, the conductance can be repeatedly switched between the two values. The conductance of the two conformations displays weak length dependencies, as is expected for guanine-rich sequences, and can be fit with a coherence-corrected hopping model. These results are supported by ab initio electronic structure calculations that indicate that the highest occupied molecular orbital is more disperse in the A-form DNA case. These results demonstrate that DNA can behave as a promising molecular switch for molecular electronics applications and also provide additional insights into the huge dispersion of DNA conductance values found in the literature.

Understanding the mechanisms of protein-DNA interactions

NASA Astrophysics Data System (ADS)

Lavery, Richard

2004-03-01

Structural, biochemical and thermodynamic data on protein-DNA interactions show that specific recognition cannot be reduced to a simple set of binary interactions between the partners (such as hydrogen bonds, ion pairs or steric contacts). The mechanical properties of the partners also play a role and, in the case of DNA, variations in both conformation and flexibility as a function of base sequence can be a significant factor in guiding a protein to the correct binding site. All-atom molecular modeling offers a means of analyzing the role of different binding mechanisms within protein-DNA complexes of known structure. This however requires estimating the binding strengths for the full range of sequences with which a given protein can interact. Since this number grows exponentially with the length of the binding site it is necessary to find a method to accelerate the calculations. We have achieved this by using a multi-copy approach (ADAPT) which allows us to build a DNA fragment with a variable base sequence. The results obtained with this method correlate well with experimental consensus binding sequences. They enable us to show that indirect recognition mechanisms involving the sequence dependent properties of DNA play a significant role in many complexes. This approach also offers a means of predicting protein binding sites on the basis of binding energies, which is complementary to conventional lexical techniques.

From Field to Laboratory: A New Database Approach for Linking Microbial Field Ecology with Laboratory Studies

NASA Technical Reports Server (NTRS)

Bebout, Leslie; Keller, R.; Miller, S.; Jahnke, L.; DeVincenzi, D. (Technical Monitor)

2002-01-01

The Ames Exobiology Culture Collection Database (AECC-DB) has been developed as a collaboration between microbial ecologists and information technology specialists. It allows for extensive web-based archiving of information regarding field samples to document microbial co-habitation of specific ecosystem micro-environments. Documentation and archiving continues as pure cultures are isolated, metabolic properties determined, and DNA extracted and sequenced. In this way metabolic properties and molecular sequences are clearly linked back to specific isolates and the location of those microbes in the ecosystem of origin. Use of this database system presents a significant advancement over traditional bookkeeping wherein there is generally little or no information regarding the environments from which microorganisms were isolated. Generally there is only a general ecosystem designation (i.e., hot-spring). However within each of these there are a myriad of microenvironments with very different properties and determining exactly where (which microenvironment) a given microbe comes from is critical in designing appropriate isolation media and interpreting physiological properties. We are currently using the database to aid in the isolation of a large number of cyanobacterial species and will present results by PI's and students demonstrating the utility of this new approach.

Next-Generation Sequencing Approaches in Genome-Wide Discovery of Single Nucleotide Polymorphism Markers Associated with Pungency and Disease Resistance in Pepper.

PubMed

Manivannan, Abinaya; Kim, Jin-Hee; Yang, Eun-Young; Ahn, Yul-Kyun; Lee, Eun-Su; Choi, Sena; Kim, Do-Sun

2018-01-01

Pepper is an economically important horticultural plant that has been widely used for its pungency and spicy taste in worldwide cuisines. Therefore, the domestication of pepper has been carried out since antiquity. Owing to meet the growing demand for pepper with high quality, organoleptic property, nutraceutical contents, and disease tolerance, genomics assisted breeding techniques can be incorporated to develop novel pepper varieties with desired traits. The application of next-generation sequencing (NGS) approaches has reformed the plant breeding technology especially in the area of molecular marker assisted breeding. The availability of genomic information aids in the deeper understanding of several molecular mechanisms behind the vital physiological processes. In addition, the NGS methods facilitate the genome-wide discovery of DNA based markers linked to key genes involved in important biological phenomenon. Among the molecular markers, single nucleotide polymorphism (SNP) indulges various benefits in comparison with other existing DNA based markers. The present review concentrates on the impact of NGS approaches in the discovery of useful SNP markers associated with pungency and disease resistance in pepper. The information provided in the current endeavor can be utilized for the betterment of pepper breeding in future.

Novel primer specific false terminations during DNA sequencing reactions: danger of inaccuracy of mutation analysis in molecular diagnostics

PubMed Central

Anwar, R; Booth, A; Churchill, A J; Markham, A F

1996-01-01

The determination of nucleotide sequence is fundamental to the identification and molecular analysis of genes. Direct sequencing of PCR products is now becoming a commonplace procedure for haplotype analysis, and for defining mutations and polymorphism within genes, particularly for diagnostic purposes. A previously unrecognised phenomenon, primer related variability, observed in sequence data generated using Taq cycle sequencing and T7 Sequenase sequencing, is reported. This suggests that caution is necessary when interpreting DNA sequence data. This is particularly important in situations where treatment may be dependent on the accuracy of the molecular diagnosis. Images PMID:16696096

Hyperthermophilic enzymes: sources, uses, and molecular mechanisms for thermostability.

PubMed

Vieille, C; Zeikus, G J

2001-03-01

Enzymes synthesized by hyperthermophiles (bacteria and archaea with optimal growth temperatures of > 80 degrees C), also called hyperthermophilic enzymes, are typically thermostable (i.e., resistant to irreversible inactivation at high temperatures) and are optimally active at high temperatures. These enzymes share the same catalytic mechanisms with their mesophilic counterparts. When cloned and expressed in mesophilic hosts, hyperthermophilic enzymes usually retain their thermal properties, indicating that these properties are genetically encoded. Sequence alignments, amino acid content comparisons, crystal structure comparisons, and mutagenesis experiments indicate that hyperthermophilic enzymes are, indeed, very similar to their mesophilic homologues. No single mechanism is responsible for the remarkable stability of hyperthermophilic enzymes. Increased thermostability must be found, instead, in a small number of highly specific alterations that often do not obey any obvious traffic rules. After briefly discussing the diversity of hyperthermophilic organisms, this review concentrates on the remarkable thermostability of their enzymes. The biochemical and molecular properties of hyperthermophilic enzymes are described. Mechanisms responsible for protein inactivation are reviewed. The molecular mechanisms involved in protein thermostabilization are discussed, including ion pairs, hydrogen bonds, hydrophobic interactions, disulfide bridges, packing, decrease of the entropy of unfolding, and intersubunit interactions. Finally, current uses and potential applications of thermophilic and hyperthermophilic enzymes as research reagents and as catalysts for industrial processes are described.

Hyperthermophilic Enzymes: Sources, Uses, and Molecular Mechanisms for Thermostability

PubMed Central

Vieille, Claire; Zeikus, Gregory J.

2001-01-01

Enzymes synthesized by hyperthermophiles (bacteria and archaea with optimal growth temperatures of >80°C), also called hyperthermophilic enzymes, are typically thermostable (i.e., resistant to irreversible inactivation at high temperatures) and are optimally active at high temperatures. These enzymes share the same catalytic mechanisms with their mesophilic counterparts. When cloned and expressed in mesophilic hosts, hyperthermophilic enzymes usually retain their thermal properties, indicating that these properties are genetically encoded. Sequence alignments, amino acid content comparisons, crystal structure comparisons, and mutagenesis experiments indicate that hyperthermophilic enzymes are, indeed, very similar to their mesophilic homologues. No single mechanism is responsible for the remarkable stability of hyperthermophilic enzymes. Increased thermostability must be found, instead, in a small number of highly specific alterations that often do not obey any obvious traffic rules. After briefly discussing the diversity of hyperthermophilic organisms, this review concentrates on the remarkable thermostability of their enzymes. The biochemical and molecular properties of hyperthermophilic enzymes are described. Mechanisms responsible for protein inactivation are reviewed. The molecular mechanisms involved in protein thermostabilization are discussed, including ion pairs, hydrogen bonds, hydrophobic interactions, disulfide bridges, packing, decrease of the entropy of unfolding, and intersubunit interactions. Finally, current uses and potential applications of thermophilic and hyperthermophilic enzymes as research reagents and as catalysts for industrial processes are described. PMID:11238984

Simultaneous optimization of biomolecular energy function on features from small molecules and macromolecules

PubMed Central

Park, Hahnbeom; Bradley, Philip; Greisen, Per; Liu, Yuan; Mulligan, Vikram Khipple; Kim, David E.; Baker, David; DiMaio, Frank

2017-01-01

Most biomolecular modeling energy functions for structure prediction, sequence design, and molecular docking, have been parameterized using existing macromolecular structural data; this contrasts molecular mechanics force fields which are largely optimized using small-molecule data. In this study, we describe an integrated method that enables optimization of a biomolecular modeling energy function simultaneously against small-molecule thermodynamic data and high-resolution macromolecular structural data. We use this approach to develop a next-generation Rosetta energy function that utilizes a new anisotropic implicit solvation model, and an improved electrostatics and Lennard-Jones model, illustrating how energy functions can be considerably improved in their ability to describe large-scale energy landscapes by incorporating both small-molecule and macromolecule data. The energy function improves performance in a wide range of protein structure prediction challenges, including monomeric structure prediction, protein-protein and protein-ligand docking, protein sequence design, and prediction of the free energy changes by mutation, while reasonably recapitulating small-molecule thermodynamic properties. PMID:27766851

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

PubMed

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

2015-12-04

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

Elucidation of Peptide-Directed Palladium Surface Structure for Biologically Tunable Nanocatalysts

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

Bedford, Nicholas M.; Ramezani-Dakhel, Hadi; Slocik, Joseph M.

Peptide-enabled synthesis of inorganic nanostructures represents an avenue to access catalytic materials with tunable and optimized properties. This is achieved via peptide complexity and programmability that is missing in traditional ligands for catalytic nanomaterials. Unfortunately, there is limited information available to correlate peptide sequence to particle structure and catalytic activity to date. As such, the application of peptide-enabled nanocatalysts remains limited to trial and error approaches. In this paper, a hybrid experimental and computational approach is introduced to systematically elucidate biomolecule-dependent structure/function relationships for peptide-capped Pd nanocatalysts. Synchrotron X-ray techniques were used to uncover substantial particle surface structural disorder, whichmore » was dependent upon the amino acid sequence of the peptide capping ligand. Nanocatalyst configurations were then determined directly from experimental data using reverse Monte Carlo methods and further refined using molecular dynamics simulation, obtaining thermodynamically stable peptide-Pd nanoparticle configurations. Sequence-dependent catalytic property differences for C-C coupling and olefin hydrogenation were then eluddated by identification of the catalytic active sites at the atomic level and quantitative prediction of relative reaction rates. This hybrid methodology provides a clear route to determine peptide-dependent structure/function relationships, enabling the generation of guidelines for catalyst design through rational tailoring of peptide sequences« less

Identification of sequence-structure RNA binding motifs for SELEX-derived aptamers.

PubMed

Hoinka, Jan; Zotenko, Elena; Friedman, Adam; Sauna, Zuben E; Przytycka, Teresa M

2012-06-15

Systematic Evolution of Ligands by EXponential Enrichment (SELEX) represents a state-of-the-art technology to isolate single-stranded (ribo)nucleic acid fragments, named aptamers, which bind to a molecule (or molecules) of interest via specific structural regions induced by their sequence-dependent fold. This powerful method has applications in designing protein inhibitors, molecular detection systems, therapeutic drugs and antibody replacement among others. However, full understanding and consequently optimal utilization of the process has lagged behind its wide application due to the lack of dedicated computational approaches. At the same time, the combination of SELEX with novel sequencing technologies is beginning to provide the data that will allow the examination of a variety of properties of the selection process. To close this gap we developed, Aptamotif, a computational method for the identification of sequence-structure motifs in SELEX-derived aptamers. To increase the chances of identifying functional motifs, Aptamotif uses an ensemble-based approach. We validated the method using two published aptamer datasets containing experimentally determined motifs of increasing complexity. We were able to recreate the author's findings to a high degree, thus proving the capability of our approach to identify binding motifs in SELEX data. Additionally, using our new experimental dataset, we illustrate the application of Aptamotif to elucidate several properties of the selection process.

De novo transcriptome sequence assembly and identification of AP2/ERF transcription factor related to abiotic stress in parsley (Petroselinum crispum).

PubMed

Li, Meng-Yao; Tan, Hua-Wei; Wang, Feng; Jiang, Qian; Xu, Zhi-Sheng; Tian, Chang; Xiong, Ai-Sheng

2014-01-01

Parsley is an important biennial Apiaceae species that is widely cultivated as herb, spice, and vegetable. Previous studies on parsley principally focused on its physiological and biochemical properties, including phenolic compound and volatile oil contents. However, little is known about the molecular and genetic properties of parsley. In this study, 23,686,707 high-quality reads were obtained and assembled into 81,852 transcripts and 50,161 unigenes for the first time. Functional annotation showed that 30,516 unigenes had sequence similarity to known genes. In addition, 3,244 putative simple sequence repeats were detected in curly parsley. Finally, 1,569 of the identified unigenes belonged to 58 transcription factor families. Various abiotic stresses have a strong detrimental effect on the yield and quality of parsley. AP2/ERF transcription factors have important functions in plant development, hormonal regulation, and abiotic response. A total of 88 putative AP2/ERF factors were identified from the transcriptome sequence of parsley. Seven AP2/ERF transcription factors were selected in this study to analyze the expression profiles of parsley under different abiotic stresses. Our data provide a potentially valuable resource that can be used for intensive parsley research.

De Novo Transcriptome Sequence Assembly and Identification of AP2/ERF Transcription Factor Related to Abiotic Stress in Parsley (Petroselinum crispum)

PubMed Central

Wang, Feng; Jiang, Qian; Xu, Zhi-Sheng; Tian, Chang; Xiong, Ai-Sheng

2014-01-01

Parsley is an important biennial Apiaceae species that is widely cultivated as herb, spice, and vegetable. Previous studies on parsley principally focused on its physiological and biochemical properties, including phenolic compound and volatile oil contents. However, little is known about the molecular and genetic properties of parsley. In this study, 23,686,707 high-quality reads were obtained and assembled into 81,852 transcripts and 50,161 unigenes for the first time. Functional annotation showed that 30,516 unigenes had sequence similarity to known genes. In addition, 3,244 putative simple sequence repeats were detected in curly parsley. Finally, 1,569 of the identified unigenes belonged to 58 transcription factor families. Various abiotic stresses have a strong detrimental effect on the yield and quality of parsley. AP2/ERF transcription factors have important functions in plant development, hormonal regulation, and abiotic response. A total of 88 putative AP2/ERF factors were identified from the transcriptome sequence of parsley. Seven AP2/ERF transcription factors were selected in this study to analyze the expression profiles of parsley under different abiotic stresses. Our data provide a potentially valuable resource that can be used for intensive parsley research. PMID:25268141

Elucidation of peptide-directed palladium surface structure for biologically tunable nanocatalysts.

PubMed

Bedford, Nicholas M; Ramezani-Dakhel, Hadi; Slocik, Joseph M; Briggs, Beverly D; Ren, Yang; Frenkel, Anatoly I; Petkov, Valeri; Heinz, Hendrik; Naik, Rajesh R; Knecht, Marc R

2015-05-26

Peptide-enabled synthesis of inorganic nanostructures represents an avenue to access catalytic materials with tunable and optimized properties. This is achieved via peptide complexity and programmability that is missing in traditional ligands for catalytic nanomaterials. Unfortunately, there is limited information available to correlate peptide sequence to particle structure and catalytic activity to date. As such, the application of peptide-enabled nanocatalysts remains limited to trial and error approaches. In this paper, a hybrid experimental and computational approach is introduced to systematically elucidate biomolecule-dependent structure/function relationships for peptide-capped Pd nanocatalysts. Synchrotron X-ray techniques were used to uncover substantial particle surface structural disorder, which was dependent upon the amino acid sequence of the peptide capping ligand. Nanocatalyst configurations were then determined directly from experimental data using reverse Monte Carlo methods and further refined using molecular dynamics simulation, obtaining thermodynamically stable peptide-Pd nanoparticle configurations. Sequence-dependent catalytic property differences for C-C coupling and olefin hydrogenation were then elucidated by identification of the catalytic active sites at the atomic level and quantitative prediction of relative reaction rates. This hybrid methodology provides a clear route to determine peptide-dependent structure/function relationships, enabling the generation of guidelines for catalyst design through rational tailoring of peptide sequences.

Sequence and properties of HMW subunit 1Bx20 from pasta wheat (Triticum durum) which is associated with poor end use properties.

PubMed

Shewry, P R; Gilbert, S M; Savage, A W J; Tatham, A S; Wan, Y-F; Belton, P S; Wellner, N; D'Ovidio, R; Békés, F; Halford, N G

2003-02-01

The gene encoding high-molecular-weight (HMW) subunit 1Bx20 was isolated from durum wheat cv. Lira. It encodes a mature protein of 774 amino acid residues with an M(r) of 83,913. Comparison with the sequence of subunit 1Bx7 showed over 96% identity, the main difference being the substitution of two cysteine residues in the N-terminal domain of subunit 1Bx7 with tyrosine residues in 1Bx20. Comparison of the structures and stabilities of the two subunits purified from wheat using Fourier-transform infra-red and circular dichroism spectroscopy showed no significant differences. However, incorporation of subunit 1Bx7 into a base flour gave increased dough strength and stability measured by Mixograph analysis, while incorporation of subunit 1Bx20 resulted in small positive or negative effects on the parameters measured. It is concluded that the different effects of the two subunits could relate to the differences in their cysteine contents, thereby affecting the cross-linking and hence properties of the glutenin polymers.

Targeted next generation sequencing for the detection of ciprofloxacin resistance markers using molecular inversion probes

DTIC Science & Technology

2016-07-06

1 Targeted next-generation sequencing for the detection of ciprofloxacin resistance markers using molecular inversion probes Christopher P...development and evaluation of a panel of 44 single-stranded molecular inversion probes (MIPs) coupled to next-generation sequencing (NGS) for the...padlock and molecular inversion probes as upfront enrichment steps for use with NGS showed the specificity and multiplexability of these techniques

The ALMA Spectroscopic Survey in the Hubble Ultra Deep Field: Molecular Gas Reservoirs in High-redshift Galaxies

NASA Astrophysics Data System (ADS)

Decarli, Roberto; Walter, Fabian; Aravena, Manuel; Carilli, Chris; Bouwens, Rychard; da Cunha, Elisabete; Daddi, Emanuele; Elbaz, David; Riechers, Dominik; Smail, Ian; Swinbank, Mark; Weiss, Axel; Bacon, Roland; Bauer, Franz; Bell, Eric F.; Bertoldi, Frank; Chapman, Scott; Colina, Luis; Cortes, Paulo C.; Cox, Pierre; Gónzalez-López, Jorge; Inami, Hanae; Ivison, Rob; Hodge, Jacqueline; Karim, Alex; Magnelli, Benjamin; Ota, Kazuaki; Popping, Gergö; Rix, Hans-Walter; Sargent, Mark; van der Wel, Arjen; van der Werf, Paul

2016-12-01

We study the molecular gas properties of high-z galaxies observed in the ALMA Spectroscopic Survey (ASPECS) that targets an ˜1 arcmin2 region in the Hubble Ultra Deep Field (UDF), a blind survey of CO emission (tracing molecular gas) in the 3 and 1 mm bands. Of a total of 1302 galaxies in the field, 56 have spectroscopic redshifts and correspondingly well-defined physical properties. Among these, 11 have infrared luminosities {L}{IR}\\gt {10}11 {L}⊙ , I.e., a detection in CO emission was expected. Out of these, 7 are detected at various significance in CO, and 4 are undetected in CO emission. In the CO-detected sources, we find CO excitation conditions that are lower than those typically found in starburst/sub-mm galaxy/QSO environments. We use the CO luminosities (including limits for non-detections) to derive molecular gas masses. We discuss our findings in the context of previous molecular gas observations at high redshift (star formation law, gas depletion times, gas fractions): the CO-detected galaxies in the UDF tend to reside on the low-{L}{IR} envelope of the scatter in the {L}{IR}{--}{L}{CO}\\prime relation, but exceptions exist. For the CO-detected sources, we find an average depletion time of ˜1 Gyr, with significant scatter. The average molecular-to-stellar mass ratio ({M}{{H}2}/M *) is consistent with earlier measurements of main-sequence galaxies at these redshifts, and again shows large variations among sources. In some cases, we also measure dust continuum emission. On average, the dust-based estimates of the molecular gas are a factor ˜2-5× smaller than those based on CO. When we account for detections as well as non-detections, we find large diversity in the molecular gas properties of the high-redshift galaxies covered by ASPECS.

Morphological and molecular characterization of a marine fish trypanosome from South Africa, including its development in a leech vector.

PubMed

Hayes, Polly M; Lawton, Scott P; Smit, Nico J; Gibson, Wendy C; Davies, Angela J

2014-01-24

Trypanosomes are ubiquitous blood parasites of marine and freshwater fishes, typically transmitted by aquatic leeches. Phylogenetic studies have been dominated by examples derived from freshwater fishes, with few marine representatives. Furthermore, life cycle studies on marine fish trypanosomes have focused on those of the northern hemisphere. In this investigation, we have examined the life cycle and molecular taxonomy of a marine fish trypanosome from South Africa. To locate trypanosome stages, leeches were removed from fishes captured on the west and south coasts of South Africa, and fish blood films and leech squashes were Giemsa-stained and screened; leeches were also examined histologically. To determine whether trypanosome stages in fishes and leeches were of the same genotype, DNA was extracted from Giemsa-stained fish blood films and leech squashes, and from fish whole blood. Fragments of the 18S rRNA gene were amplified by PCR using trypanosome-specific primers and sequenced. Resulting sequence data were compared with each other and with published trypanosome 18S rDNA sequences, and used for phylogenetic analysis. Trypanosomes were detected in blood films from fishes of the families Clinidae, Blenniidae and Gobiidae. The flagellates ranged in size and staining properties within the films and across fish hosts. In squashes and histological sections of adult and juvenile leeches, identified as Zeylanicobdella arugamensis, trypanosome developmental stages were predominantly slender epimastigotes. Sequence data showed that trypanosomes derived from fishes were identical, irrespective of whether they were small or large forms; sequences derived largely from leech epimastigotes were also identical to those obtained from fish trypanosomes. Fish and leech trypanosome sequences fell into a marine fish aquatic clade, and aligned most closely with two trypanosome sequences from marine fishes off Norway. Combined morphological and molecular methods indicate that the trypanosomes examined here represent a single pleomorphic species, rather than the three species described originally. This species is identified as Trypanosoma nudigobii Fantham, 1919 with the leech Z. arugamensis as its vector, and T. capigobii Fantham, 1919 and T. blenniclini Fantham, 1930 are regarded as junior synonyms of the species. Phylogenetic analysis establishes its affinity with marine fish trypanosomes off Norway.

Protein Modeling and Molecular Dynamics Simulation of Cloned Regucalcin (RGN) Gene from Bubalus bubalis.

PubMed

Pillai, Harikrishna; Yadav, Brijesh Singh; Chaturvedi, Navaneet; Jan, Arif Tasleem; Gupta, Girish Kumar; Baig, Mohammad Hassan; Bhure, Sanjeev Kumar

2017-01-01

Regucalcin (RGN), a calcium regulating protein having anti-prolific, antiapoptotic functions, plays important part in the biosynthesis of ascorbic acid. It is a highly conserved protein that has been reported from many tissue types of various vertebrate species. Employing its effect of regulating enzyme activities through reaction with sulfhydryl group (-SH) and calcium, structural level study believed to offer a better understanding of binding properties and regulatory mechanisms of RGN, was performed. Using sample from testis of Bubalus bubalis, amplification of regucalcin (RGN) gene was subjected to characterization by performing digestion using different restriction endonucleases (RE). Alongside, cDNA was cloned into pPICZαC vector and transformed in DH5α host for custom sequencing. To get a better insight of its structural characteristics, three dimensional (3D) structure of protein sequence was generated using in silico molecular modelling approach. The full trajectory analysis of structure was achieved by the Molecular Dynamics (MD) that explains the stability, flexibility and robustness of protein during simulation in a time of 50ns. Molecular docking against 1,5-anhydrosorbitol was performed for functional characterization of RGN. Preliminary screening of amplified products on Agarose gel showed expected size of ~893 bp of PCR product corresponding to RGN. Following sequencing, BLASTp search of the target sequence revealed that it shares 91% similarity score with human senescence marker protein-30 (pdb id: 3G4E). Molecular docking of 1,5-anhydrosorbitol reveals information regarding important binding site residues of RGN. 1,5-anhydrosorbitol was found to interact with binding free energy of - 6.01 Kcal/mol. RMSD calculation of subunits A, B and D-F might be responsible for functional and conserved regions of modeled protein. Three dimensional structure of RGN was generated and its interactions with 1,5- anhydrosorbitol, demonstrates the role of key binding residues. Until now, no structural details were available for buffalo RGN proteins, hence this study will broaden the horizon towards understanding the structural and functional aspects of different proteins in cattle. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

Inference of Markovian properties of molecular sequences from NGS data and applications to comparative genomics.

PubMed

Ren, Jie; Song, Kai; Deng, Minghua; Reinert, Gesine; Cannon, Charles H; Sun, Fengzhu

2016-04-01

Next-generation sequencing (NGS) technologies generate large amounts of short read data for many different organisms. The fact that NGS reads are generally short makes it challenging to assemble the reads and reconstruct the original genome sequence. For clustering genomes using such NGS data, word-count based alignment-free sequence comparison is a promising approach, but for this approach, the underlying expected word counts are essential.A plausible model for this underlying distribution of word counts is given through modeling the DNA sequence as a Markov chain (MC). For single long sequences, efficient statistics are available to estimate the order of MCs and the transition probability matrix for the sequences. As NGS data do not provide a single long sequence, inference methods on Markovian properties of sequences based on single long sequences cannot be directly used for NGS short read data. Here we derive a normal approximation for such word counts. We also show that the traditional Chi-square statistic has an approximate gamma distribution ,: using the Lander-Waterman model for physical mapping. We propose several methods to estimate the order of the MC based on NGS reads and evaluate those using simulations. We illustrate the applications of our results by clustering genomic sequences of several vertebrate and tree species based on NGS reads using alignment-free sequence dissimilarity measures. We find that the estimated order of the MC has a considerable effect on the clustering results ,: and that the clustering results that use a N: MC of the estimated order give a plausible clustering of the species. Our implementation of the statistics developed here is available as R package 'NGS.MC' at http://www-rcf.usc.edu/∼fsun/Programs/NGS-MC/NGS-MC.html fsun@usc.edu Supplementary data are available at Bioinformatics online. © The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

The structural genes for three Drosophila glue proteins reside at a single polytene chromosome puff locus.

PubMed Central

Crowley, T E; Bond, M W; Meyerowitz, E M

1983-01-01

The polytene chromosome puff at 68C on the Drosophila melanogaster third chromosome is thought from genetic experiments to contain the structural gene for one of the secreted salivary gland glue polypeptides, sgs-3. Previous work has demonstrated that the DNA included in this puff contains sequences that are transcribed to give three different polyadenylated RNAs that are abundant in third-larval-instar salivary glands. These have been called the group II, group III, and group IV RNAs. In the experiments reported here, we used the nucleotide sequence of the DNA coding for these RNAs to predict some of the physical and chemical properties expected of their protein products, including molecular weight, amino acid composition, and amino acid sequence. Salivary gland polypeptides with molecular weights similar to those expected for the 68C RNA translation products, and with the expected degree of incorporation of different radioactive amino acids, were purified. These proteins were shown by amino acid sequencing to correspond to the protein products of the 68C RNAs. It was further shown that each of these proteins is a part of the secreted salivary gland glue: the group IV RNA codes for the previously described sgs-3, whereas the group II and III RNAs code for the newly identified glue polypeptides sgs-8 and sgs-7. Images PMID:6406838

Sequence Directionality Dramatically Affects LCST Behavior of Elastin-Like Polypeptides.

PubMed

Li, Nan K; Roberts, Stefan; Quiroz, Felipe Garcia; Chilkoti, Ashutosh; Yingling, Yaroslava G

2018-04-30

Elastin-like polypeptides (ELP) exhibit an inverse temperature transition or lower critical solution temperature (LCST) transition phase behavior in aqueous solutions. In this paper, the thermal responsive properties of the canonical ELP, poly(VPGVG), and its reverse sequence poly(VGPVG) were investigated by turbidity measurements of the cloud point behavior, circular dichroism (CD) measurements, and all-atom molecular dynamics (MD) simulations to gain a molecular understanding of mechanism that controls hysteretic phase behavior. It was shown experimentally that both poly(VPGVG) and poly(VGPVG) undergo a transition from soluble to insoluble in aqueous solution upon heating above the transition temperature ( T t ). However, poly(VPGVG) resolubilizes upon cooling below its T t , whereas the reverse sequence, poly(VGPVG), remains aggregated despite significant undercooling below the T t . The results from MD simulations indicated that a change in sequence order results in significant differences in the dynamics of the specific residues, especially valines, which lead to extensive changes in the conformations of VPGVG and VGPVG pentamers and, consequently, dissimilar propensities for secondary structure formation and overall structure of polypeptides. These changes affected the relative hydrophilicities of polypeptides above T t , where poly(VGPVG) is more hydrophilic than poly(VPGVG) with more extended conformation and larger surface area, which led to formation of strong interchain hydrogen bonds responsible for stabilization of the aggregated phase and the observed thermal hysteresis for poly(VGPVG).

Draft genome sequence of an inbred line of Chenopodium quinoa, an allotetraploid crop with great environmental adaptability and outstanding nutritional properties.

PubMed

Yasui, Yasuo; Hirakawa, Hideki; Oikawa, Tetsuo; Toyoshima, Masami; Matsuzaki, Chiaki; Ueno, Mariko; Mizuno, Nobuyuki; Nagatoshi, Yukari; Imamura, Tomohiro; Miyago, Manami; Tanaka, Kojiro; Mise, Kazuyuki; Tanaka, Tsutomu; Mizukoshi, Hiroharu; Mori, Masashi; Fujita, Yasunari

2016-12-01

Chenopodium quinoa Willd. (quinoa) originated from the Andean region of South America, and is a pseudocereal crop of the Amaranthaceae family. Quinoa is emerging as an important crop with the potential to contribute to food security worldwide and is considered to be an optimal food source for astronauts, due to its outstanding nutritional profile and ability to tolerate stressful environments. Furthermore, plant pathologists use quinoa as a representative diagnostic host to identify virus species. However, molecular analysis of quinoa is limited by its genetic heterogeneity due to outcrossing and its genome complexity derived from allotetraploidy. To overcome these obstacles, we established the inbred and standard quinoa accession Kd that enables rigorous molecular analysis, and presented the draft genome sequence of Kd, using an optimized combination of high-throughput next generation sequencing on the Illumina Hiseq 2500 and PacBio RS II sequencers. The de novo genome assembly contained 25 k scaffolds consisting of 1 Gbp with N50 length of 86 kbp. Based on these data, we constructed the free-access Quinoa Genome DataBase (QGDB). Thus, these findings provide insights into the mechanisms underlying agronomically important traits of quinoa and the effect of allotetraploidy on genome evolution. © The Author 2016. Published by Oxford University Press on behalf of Kazusa DNA Research Institute.

Molecular and immunological characterisation of the glycosylated orange allergen Cit s 1

PubMed Central

Pöltl, Gerald; Ahrazem, Oussama; Paschinger, Katharina; Ibañez, M. Dolores; Salcedo, Gabriel; Wilson, Iain B. H.

2010-01-01

The IgE of sera from patients with a history of allergy to oranges (Citrus sinensis) bind a number of proteins in orange extract, including Cit s 1, a germin-like protein. In the present study, we have analysed its immunological cross-reactivity and its molecular nature. Sera from many of the patients examined recognise a range of glycoproteins and neoglycoconjugates containing β1,2-xylose and core α1,3-fucose on their N-glycans. These reagents also inhibited the interaction of Cit s 1 with patients’ sera, thus underlining the critical role of glycosylation in the recognition of this protein by patients’ IgE and extending previous data showing that deglycosylated Cit s 1 does not possess IgE epitopes. In parallel, we examined the peptide sequence and glycan structure of Cit s 1 using mass spectrometric techniques. Indeed, we achieved complete sequence coverage of the mature protein as compared to the translation of an expressed sequence tag cDNA clone and demonstrated that the single N-glycosylation site of this protein carries oligosaccharides with xylose and fucose residues. Due to the presumed requirement for multivalency for in vivo allergenicity, our molecular data showing that Cit s 1 is monovalent as regards glycosylation and that the single N-glycan is the target of the IgE response to this protein, therefore, explain the immunological cross-reactive properties of Cit s 1 as well as its equivocal nature as a clinically-relevant allergen. PMID:17095532

Transcriptome analysis of pecan seeds at different developing stages and identification of key genes involved in lipid metabolism

PubMed Central

Shah, Faheem Afzal; Wang, Qiaojian; Wang, Zhaocheng; Wu, Lifang

2018-01-01

Pecan is an economically important nut crop tree due to its unique texture and flavor properties. The pecan seed is rich of unsaturated fatty acid and protein. However, little is known about the molecular mechanisms of the biosynthesis of fatty acids in the developing seeds. In this study, transcriptome sequencing of the developing seeds was performed using Illumina sequencing technology. Pecan seed embryos at different developmental stages were collected and sequenced. The transcriptomes of pecan seeds at two key developing stages (PA, the initial stage and PS, the fast oil accumulation stage) were also compared. A total of 82,155 unigenes, with an average length of 1,198 bp from seven independent libraries were generated. After functional annotations, we detected approximately 55,854 CDS, among which, 2,807 were Transcription Factor (TF) coding unigenes. Further, there were 13,325 unigenes that showed a 2-fold or greater expression difference between the two groups of libraries (two developmental stages). After transcriptome analysis, we identified abundant unigenes that could be involved in fatty acid biosynthesis, degradation and some other aspects of seed development in pecan. This study presents a comprehensive dataset of transcriptomic changes during the seed development of pecan. It provides insights in understanding the molecular mechanisms responsible for fatty acid biosynthesis in the seed development. The identification of functional genes will also be useful for the molecular breeding work of pecan. PMID:29694395

Transcriptome analysis of pecan seeds at different developing stages and identification of key genes involved in lipid metabolism.

PubMed

Xu, Zheng; Ni, Jun; Shah, Faheem Afzal; Wang, Qiaojian; Wang, Zhaocheng; Wu, Lifang; Fu, Songling

2018-01-01

Pecan is an economically important nut crop tree due to its unique texture and flavor properties. The pecan seed is rich of unsaturated fatty acid and protein. However, little is known about the molecular mechanisms of the biosynthesis of fatty acids in the developing seeds. In this study, transcriptome sequencing of the developing seeds was performed using Illumina sequencing technology. Pecan seed embryos at different developmental stages were collected and sequenced. The transcriptomes of pecan seeds at two key developing stages (PA, the initial stage and PS, the fast oil accumulation stage) were also compared. A total of 82,155 unigenes, with an average length of 1,198 bp from seven independent libraries were generated. After functional annotations, we detected approximately 55,854 CDS, among which, 2,807 were Transcription Factor (TF) coding unigenes. Further, there were 13,325 unigenes that showed a 2-fold or greater expression difference between the two groups of libraries (two developmental stages). After transcriptome analysis, we identified abundant unigenes that could be involved in fatty acid biosynthesis, degradation and some other aspects of seed development in pecan. This study presents a comprehensive dataset of transcriptomic changes during the seed development of pecan. It provides insights in understanding the molecular mechanisms responsible for fatty acid biosynthesis in the seed development. The identification of functional genes will also be useful for the molecular breeding work of pecan.

Supramolecular Architectures and Mimics of Complex Natural Folds Derived from Rationally Designed alpha-Helical Protein Structures

NASA Astrophysics Data System (ADS)

Tavenor, Nathan Albert

Protein-based supramolecular polymers (SMPs) are a class of biomaterials which draw inspiration from and expand upon the many examples of complex protein quaternary structures observed in nature: collagen, microtubules, viral capsids, etc. Designing synthetic supramolecular protein scaffolds both increases our understanding of natural superstructures and allows for the creation of novel materials. Similar to small-molecule SMPs, protein-based SMPs form due to self-assembly driven by intermolecular interactions between monomers, and monomer structure determines the properties of the overall material. Using protein-based monomers takes advantage of the self-assembly and highly specific molecular recognition properties encodable in polypeptide sequences to rationally design SMP architectures. The central hypothesis underlying our work is that alpha-helical coiled coils, a well-studied protein quaternary folding motif, are well-suited to SMP design through the addition of synthetic linkers at solvent-exposed sites. Through small changes in the structures of the cross-links and/or peptide sequence, we have been able to control both the nanoscale organization and the macroscopic properties of the SMPs. Changes to the linker and hydrophobic core of the peptide can be used to control polymer rigidity, stability, and dimensionality. The gaps in knowledge that this thesis sought to fill on this project were 1) the relationship between the molecular structure of the cross-linked polypeptides and the macroscopic properties of the SMPs and 2) a means of creating materials exhibiting multi-dimensional net or framework topologies. Separate from the above efforts on supramolecular architectures was work on improving backbone modification strategies for an alpha-helix in the context of a complex protein tertiary fold. Earlier work in our lab had successfully incorporated unnatural building blocks into every major secondary structure (beta-sheet, alpha-helix, loops and beta-turns) of a small protein with a tertiary fold. Although the tertiary fold of the native sequence was mimicked by the resulting artificial protein, the thermodynamic stability was greatly compromised. Most of this energetic penalty derived from the modifications present in the alpha-helix. The contribution within this thesis was direct comparison of several alpha-helical design strategies and establishment of the thermodynamic consequences of each.

Generation and analysis of ESTs from strawberry (Fragaria xananassa) fruits and evaluation of their utility in genetic and molecular studies

PubMed Central

2010-01-01

Background Cultivated strawberry is a hybrid octoploid species (Fragaria xananassa Duchesne ex. Rozier) whose fruit is highly appreciated due to its organoleptic properties and health benefits. Despite recent studies on the control of its growth and ripening processes, information about the role played by different hormones on these processes remains elusive. Further advancement of this knowledge is hampered by the limited sequence information on genes from this species, despite the abundant information available on genes from the wild diploid relative Fragaria vesca. However, the diploid species, or one ancestor, only partially contributes to the genome of the cultivated octoploid. We have produced a collection of expressed sequence tags (ESTs) from different cDNA libraries prepared from different fruit parts and developmental stages. The collection has been analysed and the sequence information used to explore the involvement of different hormones in fruit developmental processes, and for the comparison of transcripts in the receptacle of ripe fruits of diploid and octoploid species. The study is particularly important since the commercial fruit is indeed an enlarged flower receptacle with the true fruits, the achenes, on the surface and connected through a network of vascular vessels to the central pith. Results We have sequenced over 4,500 ESTs from Fragaria xananassa, thus doubling the number of ESTs available in the GenBank of this species. We then assembled this information together with that available from F. xananassa resulting a total of 7,096 unigenes. The identification of SSRs and SNPs in many of the ESTs allowed their conversion into functional molecular markers. The availability of libraries prepared from green growing fruits has allowed the cloning of cDNAs encoding for genes of auxin, ethylene and brassinosteroid signalling processes, followed by expression studies in selected fruit parts and developmental stages. In addition, the sequence information generated in the project, jointly with previous information on sequences from both F. xananassa and F. vesca, has allowed designing an oligo-based microarray that has been used to compare the transcriptome of the ripe receptacle of the diploid and octoploid species. Comparison of the transcriptomes, grouping the genes by biological processes, points to differences being quantitative rather than qualitative. Conclusions The present study generates essential knowledge and molecular tools that will be useful in improving investigations at the molecular level in cultivated strawberry (F. xananassa). This knowledge is likely to provide useful resources in the ongoing breeding programs. The sequence information has already allowed the development of molecular markers that have been applied to germplasm characterization and could be eventually used in QTL analysis. Massive transcription analysis can be of utility to target specific genes to be further studied, by their involvement in the different plant developmental processes. PMID:20849591

Sequence of a cDNA encoding pancreatic preprosomatostatin-22.

PubMed Central

Magazin, M; Minth, C D; Funckes, C L; Deschenes, R; Tavianini, M A; Dixon, J E

1982-01-01

We report the nucleotide sequence of a precursor to somatostatin that upon proteolytic processing may give rise to a hormone of 22 amino acids. The nucleotide sequence of a cDNA from the channel catfish (Ictalurus punctatus) encodes a precursor to somatostatin that is 105 amino acids (Mr, 11,500). The cDNA coding for somatostatin-22 consists of 36 nucleotides in the 5' untranslated region, 315 nucleotides that code for the precursor to somatostatin-22, 269 nucleotides at the 3' untranslated region, and a variable length of poly(A). The putative preprohormone contains a sequence of hydrophobic amino acids at the amino terminus that has the properties of a "signal" peptide. A connecting sequence of approximately 57 amino acids is followed by a single Arg-Arg sequence, which immediately precedes the hormone. Somatostatin-22 is homologous to somatostatin-14 in 7 of the 14 amino acids, including the Phe-Trp-Lys sequence. Hybridization selection of mRNA, followed by its translation in a wheat germ cell-free system, resulted in the synthesis of a single polypeptide having a molecular weight of approximately 10,000 as estimated on Na-DodSO4/polyacrylamide gels. Images PMID:6127673

Information management in DNA replication modeled by directional, stochastic chains with memory

NASA Astrophysics Data System (ADS)

Arias-Gonzalez, J. Ricardo

2016-11-01

Stochastic chains represent a key variety of phenomena in many branches of science within the context of information theory and thermodynamics. They are typically approached by a sequence of independent events or by a memoryless Markov process. Stochastic chains are of special significance to molecular biology, where genes are conveyed by linear polymers made up of molecular subunits and transferred from DNA to proteins by specialized molecular motors in the presence of errors. Here, we demonstrate that when memory is introduced, the statistics of the chain depends on the mechanism by which objects or symbols are assembled, even in the slow dynamics limit wherein friction can be neglected. To analyze these systems, we introduce a sequence-dependent partition function, investigate its properties, and compare it to the standard normalization defined by the statistical physics of ensembles. We then apply this theory to characterize the enzyme-mediated information transfer involved in DNA replication under the real, non-equilibrium conditions, reproducing measured error rates and explaining the typical 100-fold increase in fidelity that is experimentally found when proofreading and edition take place. Our model further predicts that approximately 1 kT has to be consumed to elevate fidelity in one order of magnitude. We anticipate that our results are necessary to interpret configurational order and information management in many molecular systems within biophysics, materials science, communication, and engineering.

C. elegans network biology: a beginning.

PubMed Central

Piano, Fabio; Gunsalus, Kristin C; Hill, David E; Vidal, Marc

2006-01-01

The architecture and dynamics of molecular networks can provide an understanding of complex biological processes complementary to that obtained from the in-depth study of single genes and proteins. With a completely sequenced and well-annotated genome, a fully characterized cell lineage, and powerful tools available to dissect development, Caenorhabditis elegans, among metazoans, provides an optimal system to bridge cellular and organismal biology with the global properties of macromolecular networks. This chapter considers omic technologies available for C. elegans to describe molecular networks--encompassing transcriptional and phenotypic profiling as well as physical interaction mapping--and discusses how their individual and integrated applications are paving the way for a network-level understanding of C. elegans biology. PMID:18050437

Study design requirements for RNA sequencing-based breast cancer diagnostics.

PubMed

Mer, Arvind Singh; Klevebring, Daniel; Grönberg, Henrik; Rantalainen, Mattias

2016-02-01

Sequencing-based molecular characterization of tumors provides information required for individualized cancer treatment. There are well-defined molecular subtypes of breast cancer that provide improved prognostication compared to routine biomarkers. However, molecular subtyping is not yet implemented in routine breast cancer care. Clinical translation is dependent on subtype prediction models providing high sensitivity and specificity. In this study we evaluate sample size and RNA-sequencing read requirements for breast cancer subtyping to facilitate rational design of translational studies. We applied subsampling to ascertain the effect of training sample size and the number of RNA sequencing reads on classification accuracy of molecular subtype and routine biomarker prediction models (unsupervised and supervised). Subtype classification accuracy improved with increasing sample size up to N = 750 (accuracy = 0.93), although with a modest improvement beyond N = 350 (accuracy = 0.92). Prediction of routine biomarkers achieved accuracy of 0.94 (ER) and 0.92 (Her2) at N = 200. Subtype classification improved with RNA-sequencing library size up to 5 million reads. Development of molecular subtyping models for cancer diagnostics requires well-designed studies. Sample size and the number of RNA sequencing reads directly influence accuracy of molecular subtyping. Results in this study provide key information for rational design of translational studies aiming to bring sequencing-based diagnostics to the clinic.

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

PubMed

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

2013-12-01

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

Variation in Orthologous Shell-Forming Proteins Contribute to Molluscan Shell Diversity.

PubMed

Jackson, Daniel J; Reim, Laurin; Randow, Clemens; Cerveau, Nicolas; Degnan, Bernard M; Fleck, Claudia

2017-11-01

Despite the evolutionary success and ancient heritage of the molluscan shell, little is known about the molecular details of its formation, evolutionary origins, or the interactions between the material properties of the shell and its organic constituents. In contrast to this dearth of information, a growing collection of molluscan shell-forming proteomes and transcriptomes suggest they are comprised of both deeply conserved, and lineage specific elements. Analyses of these sequence data sets have suggested that mechanisms such as exon shuffling, gene co-option, and gene family expansion facilitated the rapid evolution of shell-forming proteomes and supported the diversification of this phylum specific structure. In order to further investigate and test these ideas we have examined the molecular features and spatial expression patterns of two shell-forming genes (Lustrin and ML1A2) and coupled these observations with materials properties measurements of shells from a group of closely related gastropods (abalone). We find that the prominent "GS" domain of Lustrin, a domain believed to confer elastomeric properties to the shell, varies significantly in length between the species we investigated. Furthermore, the spatial expression patterns of Lustrin and ML1A2 also vary significantly between species, suggesting that both protein architecture, and the regulation of spatial gene expression patterns, are important drivers of molluscan shell evolution. Variation in these molecular features might relate to certain materials properties of the shells of these species. These insights reveal an important and underappreciated source of variation within shell-forming proteomes that must contribute to the diversity of molluscan shell phenotypes. © The Author 2017. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.

Binding properties of SUMO-interacting motifs (SIMs) in yeast.

PubMed

Jardin, Christophe; Horn, Anselm H C; Sticht, Heinrich

2015-03-01

Small ubiquitin-like modifier (SUMO) conjugation and interaction play an essential role in many cellular processes. A large number of yeast proteins is known to interact non-covalently with SUMO via short SUMO-interacting motifs (SIMs), but the structural details of this interaction are yet poorly characterized. In the present work, sequence analysis of a large dataset of 148 yeast SIMs revealed the existence of a hydrophobic core binding motif and a preference for acidic residues either within or adjacent to the core motif. Thus the sequence properties of yeast SIMs are highly similar to those described for human. Molecular dynamics simulations were performed to investigate the binding preferences for four representative SIM peptides differing in the number and distribution of acidic residues. Furthermore, the relative stability of two previously observed alternative binding orientations (parallel, antiparallel) was assessed. For all SIMs investigated, the antiparallel binding mode remained stable in the simulations and the SIMs were tightly bound via their hydrophobic core residues supplemented by polar interactions of the acidic residues. In contrary, the stability of the parallel binding mode is more dependent on the sequence features of the SIM motif like the number and position of acidic residues or the presence of additional adjacent interaction motifs. This information should be helpful to enhance the prediction of SIMs and their binding properties in different organisms to facilitate the reconstruction of the SUMO interactome.

Evolutionary history of versatile-lipases from Agaricales through reconstruction of ancestral structures.

PubMed

Barriuso, Jorge; Martínez, María Jesús

2017-01-03

Fungal "Versatile carboxylic ester hydrolases" are enzymes with great biotechnological interest. Here we carried out a bioinformatic screening to find these proteins in genomes from Agaricales, by means of searching for conserved motifs, sequence and phylogenetic analysis, and three-dimensional modeling. Moreover, we reconstructed the molecular evolution of these enzymes along the time by inferring and analyzing the sequence of ancestral intermediate forms. The properties of the ancestral candidates are discussed on the basis of their three-dimensional structural models, the hydrophobicity of the lid, and the substrate binding intramolecular tunnel, revealing all of them featured properties of these enzymes. The evolutionary history of the putative lipases revealed an increase on the length and hydrophobicity of the lid region, as well as in the size of the substrate binding pocket, during evolution time. These facts suggest the enzymes' specialization towards certain substrates and their subsequent loss of promiscuity. These results bring to light the presence of different pools of lipases in fungi with different habitats and life styles. Despite the consistency of the data gathered from reconstruction of ancestral sequences, the heterologous expression of some of these candidates would be essential to corroborate enzymes' activities.

A Parvovirus B19 synthetic genome: sequence features and functional competence.

PubMed

Manaresi, Elisabetta; Conti, Ilaria; Bua, Gloria; Bonvicini, Francesca; Gallinella, Giorgio

2017-08-01

Central to genetic studies for Parvovirus B19 (B19V) is the availability of genomic clones that may possess functional competence and ability to generate infectious virus. In our study, we established a new model genetic system for Parvovirus B19. A synthetic approach was followed, by design of a reference genome sequence, by generation of a corresponding artificial construct and its molecular cloning in a complete and functional form, and by setup of an efficient strategy to generate infectious virus, via transfection in UT7/EpoS1 cells and amplification in erythroid progenitor cells. The synthetic genome was able to generate virus with biological properties paralleling those of native virus, its infectious activity being dependent on the preservation of self-complementarity and sequence heterogeneity within the terminal regions. A virus of defined genome sequence, obtained from controlled cell culture conditions, can constitute a reference tool for investigation of the structural and functional characteristics of the virus. Copyright © 2017 Elsevier Inc. All rights reserved.

Cloning and High-Level Expression of α-Galactosidase cDNA from Penicillium purpurogenum

PubMed Central

Shibuya, Hajime; Nagasaki, Hiroaki; Kaneko, Satoshi; Yoshida, Shigeki; Park, Gwi Gun; Kusakabe, Isao; Kobayashi, Hideyuki

1998-01-01

The cDNA coding for Penicillium purpurogenum α-galactosidase (αGal) was cloned and sequenced. The deduced amino acid sequence of the α-Gal cDNA showed that the mature enzyme consisted of 419 amino acid residues with a molecular mass of 46,334 Da. The derived amino acid sequence of the enzyme showed similarity to eukaryotic αGals from plants, animals, yeasts, and filamentous fungi. The highest similarity observed (57% identity) was to Trichoderma reesei AGLI. The cDNA was expressed in Saccharomyces cerevisiae under the control of the yeast GAL10 promoter. Almost all of the enzyme produced was secreted into the culture medium, and the expression level reached was approximately 0.2 g/liter. The recombinant enzyme purified to homogeneity was highly glycosylated, showed slightly higher specific activity, and exhibited properties almost identical to those of the native enzyme from P. purpurogenum in terms of the N-terminal amino acid sequence, thermoactivity, pH profile, and mode of action on galacto-oligosaccharides. PMID:9797312

ProteomeVis: a web app for exploration of protein properties from structure to sequence evolution across organisms' proteomes.

PubMed

Razban, Rostam M; Gilson, Amy I; Durfee, Niamh; Strobelt, Hendrik; Dinkla, Kasper; Choi, Jeong-Mo; Pfister, Hanspeter; Shakhnovich, Eugene I

2018-05-08

Protein evolution spans time scales and its effects span the length of an organism. A web app named ProteomeVis is developed to provide a comprehensive view of protein evolution in the S. cerevisiae and E. coli proteomes. ProteomeVis interactively creates protein chain graphs, where edges between nodes represent structure and sequence similarities within user-defined ranges, to study the long time scale effects of protein structure evolution. The short time scale effects of protein sequence evolution are studied by sequence evolutionary rate (ER) correlation analyses with protein properties that span from the molecular to the organismal level. We demonstrate the utility and versatility of ProteomeVis by investigating the distribution of edges per node in organismal protein chain universe graphs (oPCUGs) and putative ER determinants. S. cerevisiae and E. coli oPCUGs are scale-free with scaling constants of 1.79 and 1.56, respectively. Both scaling constants can be explained by a previously reported theoretical model describing protein structure evolution (Dokholyan et al., 2002). Protein abundance most strongly correlates with ER among properties in ProteomeVis, with Spearman correlations of -0.49 (p-value<10-10) and -0.46 (p-value<10-10) for S. cerevisiae and E. coli, respectively. This result is consistent with previous reports that found protein expression to be the most important ER determinant (Zhang and Yang, 2015). ProteomeVis is freely accessible at http://proteomevis.chem.harvard.edu. Supplementary data are available at Bioinformatics. shakhnovich@chemistry.harvard.edu.

Polymorphic phase transitions and molecular motion in pyridinium chlorochromate

NASA Astrophysics Data System (ADS)

Pajaķ, Z.; Szafrańska, B.; Czarnecki, P.; Mayer, J.; Kozak, A.

1997-08-01

DTA, DSC, NMR and dielectric studies have been performed for pyridinium chlorochromate over a wide temperature range. A sequence of four solid-solid phase transitions was discovered. The in-plane complex reorientation of the cation is described by a three-well potential model with two correlation times. At higher temperatures one observes simultaneous cation tumbling and diffusion. Thus existence of a new ionic plastic phase is revealed. The domain structure observed suggests ferroelastic properties of the compound.

Purification and properties of a beta-galactosidase from carambola fruit with significant activity towards cell wall polysaccharides.

PubMed

Balasubramaniam, Sumathi; Lee, Heng Chin; Lazan, Hamid; Othman, Roohaida; Ali, Zainon Mohd

2005-01-01

beta-Galactosidase (EC. 3.2.1.23) from ripe carambola (Averrhoa carambola L. cv. B10) fruit was fractionated through a combination of ion exchange and gel filtration chromatography into four isoforms, viz. beta-galactosidase I, II, III and IV. This beta-galactosidases had apparent native molecular masses of 84, 77, 58 and 130 kDa, respectively. beta-Galactosidase I, the predominant isoform, was purified to electrophoretic homogeneity; analysis of the protein by SDS-PAGE revealed two subunits with molecular masses of 48 and 36 kDa. N-terminal amino acid sequence of the respective polypeptides shared high similarities albeit at different domains, with the deduced amino acid sequence of certain plant beta-galactosidases, thus, explaining the observed low similarity between the two subunits. beta-Galactosidase I was probably a heterodimer that have glycoprotein properties and a pI value of 7.2, with one of the potential glycosylation sites appeared to reside within the 48-kDa-polypeptide. The purified beta-galactosidase I was substantially active in hydrolyzing (1-->4)beta-linked spruce and a mixture of (1-->3)beta- and (1-->6)beta-linked gum arabic galactans. This isoform also had the capability to solubilize and depolymerize structurally intact pectins as well as to modify alkaline-soluble hemicelluloses, reflecting in part changes that occur during ripening.

Long-range correlation properties of coding and noncoding DNA sequences: GenBank analysis.

PubMed

Buldyrev, S V; Goldberger, A L; Havlin, S; Mantegna, R N; Matsa, M E; Peng, C K; Simons, M; Stanley, H E

1995-05-01

An open question in computational molecular biology is whether long-range correlations are present in both coding and noncoding DNA or only in the latter. To answer this question, we consider all 33301 coding and all 29453 noncoding eukaryotic sequences--each of length larger than 512 base pairs (bp)--in the present release of the GenBank to dtermine whether there is any statistically significant distinction in their long-range correlation properties. Standard fast Fourier transform (FFT) analysis indicates that coding sequences have practically no correlations in the range from 10 bp to 100 bp (spectral exponent beta=0.00 +/- 0.04, where the uncertainty is two standard deviations). In contrast, for noncoding sequences, the average value of the spectral exponent beta is positive (0.16 +/- 0.05) which unambiguously shows the presence of long-range correlations. We also separately analyze the 874 coding and the 1157 noncoding sequences that have more than 4096 bp and find a larger region of power-law behavior. We calculate the probability that these two data sets (coding and noncoding) were drawn from the same distribution and we find that it is less than 10(-10). We obtain independent confirmation of these findings using the method of detrended fluctuation analysis (DFA), which is designed to treat sequences with statistical heterogeneity, such as DNA's known mosaic structure ("patchiness") arising from the nonstationarity of nucleotide concentration. The near-perfect agreement between the two independent analysis methods, FFT and DFA, increases the confidence in the reliability of our conclusion.

Long-range correlation properties of coding and noncoding DNA sequences: GenBank analysis

NASA Technical Reports Server (NTRS)

Buldyrev, S. V.; Goldberger, A. L.; Havlin, S.; Mantegna, R. N.; Matsa, M. E.; Peng, C. K.; Simons, M.; Stanley, H. E.

1995-01-01

An open question in computational molecular biology is whether long-range correlations are present in both coding and noncoding DNA or only in the latter. To answer this question, we consider all 33301 coding and all 29453 noncoding eukaryotic sequences--each of length larger than 512 base pairs (bp)--in the present release of the GenBank to dtermine whether there is any statistically significant distinction in their long-range correlation properties. Standard fast Fourier transform (FFT) analysis indicates that coding sequences have practically no correlations in the range from 10 bp to 100 bp (spectral exponent beta=0.00 +/- 0.04, where the uncertainty is two standard deviations). In contrast, for noncoding sequences, the average value of the spectral exponent beta is positive (0.16 +/- 0.05) which unambiguously shows the presence of long-range correlations. We also separately analyze the 874 coding and the 1157 noncoding sequences that have more than 4096 bp and find a larger region of power-law behavior. We calculate the probability that these two data sets (coding and noncoding) were drawn from the same distribution and we find that it is less than 10(-10). We obtain independent confirmation of these findings using the method of detrended fluctuation analysis (DFA), which is designed to treat sequences with statistical heterogeneity, such as DNA's known mosaic structure ("patchiness") arising from the nonstationarity of nucleotide concentration. The near-perfect agreement between the two independent analysis methods, FFT and DFA, increases the confidence in the reliability of our conclusion.

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

PubMed

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

2017-09-11

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

Unraveling systematic inventory of Echinops (Asteraceae) with special reference to nrDNA ITS sequence-based molecular typing of Echinops abuzinadianus.

PubMed

Ali, M A; Al-Hemaid, F M; Lee, J; Hatamleh, A A; Gyulai, G; Rahman, M O

2015-10-02

The present study explored the systematic inventory of Echinops L. (Asteraceae) of Saudi Arabia, with special reference to the molecular typing of Echinops abuzinadianus Chaudhary, an endemic species to Saudi Arabia, based on the internal transcribed spacer (ITS) sequences (ITS1-5.8S-ITS2) of nuclear ribosomal DNA. A sequence similarity search using BLAST and a phylogenetic analysis of the ITS sequence of E. abuzinadianus revealed a high level of sequence similarity with E. glaberrimus DC. (section Ritropsis). The novel primary sequence and the secondary structure of ITS2 of E. abuzinadianus could potentially be used for molecular genotyping.

[Analysis of Conformational Features of Watson-Crick Duplex Fragments by Molecular Mechanics and Quantum Mechanics Methods].

PubMed

Poltev, V I; Anisimov, V M; Sanchez, C; Deriabina, A; Gonzalez, E; Garcia, D; Rivas, F; Polteva, N A

2016-01-01

It is generally accepted that the important characteristic features of the Watson-Crick duplex originate from the molecular structure of its subunits. However, it still remains to elucidate what properties of each subunit are responsible for the significant characteristic features of the DNA structure. The computations of desoxydinucleoside monophosphates complexes with Na-ions using density functional theory revealed a pivotal role of DNA conformational properties of single-chain minimal fragments in the development of unique features of the Watson-Crick duplex. We found that directionality of the sugar-phosphate backbone and the preferable ranges of its torsion angles, combined with the difference between purines and pyrimidines. in ring bases, define the dependence of three-dimensional structure of the Watson-Crick duplex on nucleotide base sequence. In this work, we extended these density functional theory computations to the minimal' fragments of DNA duplex, complementary desoxydinucleoside monophosphates complexes with Na-ions. Using several computational methods and various functionals, we performed a search for energy minima of BI-conformation for complementary desoxydinucleoside monophosphates complexes with different nucleoside sequences. Two sequences are optimized using ab initio method at the MP2/6-31++G** level of theory. The analysis of torsion angles, sugar ring puckering and mutual base positions of optimized structures demonstrates that the conformational characteristic features of complementary desoxydinucleoside monophosphates complexes with Na-ions remain within BI ranges and become closer to the corresponding characteristic features of the Watson-Crick duplex crystals. Qualitatively, the main characteristic features of each studied complementary desoxydinucleoside monophosphates complex remain invariant when different computational methods are used, although the quantitative values of some conformational parameters could vary lying within the limits typical for the corresponding family. We observe that popular functionals in density functional theory calculations lead to the overestimated distances between base pairs, while MP2 computations and the newer complex functionals produce the structures that have too close atom-atom contacts. A detailed study of some complementary desoxydinucleoside monophosphate complexes with Na-ions highlights the existence of several energy minima corresponding to BI-conformations, in other words, the complexity of the relief pattern of the potential energy surface of complementary desoxydinucleoside monophosphate complexes. This accounts for variability of conformational parameters of duplex fragments with the same base sequence. Popular molecular mechanics force fields AMBER and CHARMM reproduce most of the conformational characteristics of desoxydinucleoside monophosphates and their complementary complexes with Na-ions but fail to reproduce some details of the dependence of the Watson-Crick duplex conformation on the nucleotide sequence.

Characterization and biological properties of a new staphylococcal exotoxin

PubMed Central

1994-01-01

Staphylococcus aureus strain D4508 is a toxic shock syndrome toxin 1- negative clinical isolate from a nonmenstrual case of toxic shock syndrome (TSS). In the present study, we have purified and characterized a new exotoxin from the extracellular products of this strain. This toxin was found to have a molecular mass of 25.14 kD by mass spectrometry and an isoelectric point of 5.65 by isoelectric focusing. We have also cloned and sequenced its corresponding genomic determinant. The DNA sequence encoding the mature protein was found to be 654 base pairs and is predicted to encode a polypeptide of 218 amino acids. The deduced protein contains an NH2-terminal sequence identical to that of the native protein. The calculated molecular weight (25.21 kD) of the recombinant mature protein is also consistent with that of the native molecules. When injected intravenously into rabbits, both the native and recombinant toxins induce an acute TSS-like illness characterized by high fever, hypotension, diarrhea, shock, and in some cases death, with classical histological findings of TSS. Furthermore, the activity of the toxin is specifically enhanced by low quantities of endotoxins. The toxicity can be blocked by rabbit immunoglobulin G antibody specific for the toxin. Western blotting and DNA sequencing data confirm that the protein is a unique staphylococcal exotoxin, yet shares significant sequence homology with known staphylococcal enterotoxins, especially the SEA, SED, and SEE toxins. We conclude therefore that this 25-kD protein belongs to the staphylococcal enterotoxin gene family that is capable of inducing a TSS-like illness in rabbits. PMID:7964453

Molecular characterization of domestic and exotic potato virus S isolates and a global analysis of genomic sequences.

PubMed

Lin, Y-H; Abad, J A; Maroon-Lango, C J; Perry, K L; Pappu, H R

2014-08-01

Five potato virus S (PVS) isolates from the USA and three isolates from Chile were characterized based on biological and molecular properties to delineate these PVS isolates into either ordinary (PVS(O)) or Andean (PVS(A)) strains. Five isolates - 41956, Cosimar, Galaxy, ND2492-2R, and Q1 - were considered ordinary strains, as they induced local lesions on the inoculated leaves of Chenopodium quinoa, whereas the remaining three (FL206-1D, Q3, and Q5) failed to induce symptoms. Considerable variability of symptom expression and severity was observed among these isolates when tested on additional indicator plants and potato cv. Defender. Additionally, all eight isolates were characterized by determining the nucleotide sequences of their coat protein (CP) genes. Based on their biological and genetic properties, the 41956, Cosimar, Galaxy, ND2492-2R, and Q1 isolates were identified as PVS(O). PVS-FL206-1D and the two Chilean isolates (PVS-Q3 and PVS-Q5) could not be identified based on phenotype alone; however, based on sequence comparisons, PVS-FL206-1D was identified as PVS(O), while Q3 and Q5 clustered with known PVS(A) strains. C. quinoa may not be a reliable indicator for distinguishing PVS strains. Sequences of the CP gene should be used as an additional criterion for delineating PVS strains. A global genetic analysis of known PVS sequences from GenBank was carried out to investigate nucleotide substitution, population selection, and genetic recombination and to assess the genetic diversity and evolution of PVS. A higher degree of nucleotide diversity (π value) of the CP gene compared to that of the 11K gene suggested greater variation in the CP gene. When comparing PVS(A) and PVS(O) strains, a higher π value was found for PVS(A). Statistical tests of the neutrality hypothesis indicated a negative selection pressure on both the CP and 11K proteins of PVS(O), whereas a balancing selection pressure was found on PVS(A).

Conjugal transfer using the bacteriophage phiC31 att/int system and properties of the attB site in Streptomyces ambofaciens.

PubMed

Kim, Mi-Kyung; Ha, Heon-Su; Choi, Sun-Uk

2008-04-01

To facilitate molecular genetic studies of Streptomyces ambofaciens that produces spiramycin, a commercially important macrolide antibiotic used in human medicine against Gram-positive pathogenic bacteria, the conditions for the conjugal transfer of DNA from E. coli to S. ambofaciens were established using a bacteriophage phiC31 att/int system. The transconjugation efficiency of S. ambofaciens varied with the medium used; the highest frequency was obtained on AS-1 medium containing 10 mM MgCl(2) without heat treatment of the spores. In addition, by cloning and sequencing the attB site, we identified that S. ambofaciens contains a single attB site within an ORF coding for a pirin homolog, and its attB site sequence shows 100% nt identity to the sequence of S. coelicolor and S. lividans, which have the highest efficiency in transconjugation using the phiC31 att/int system.

Identification of a Novel Recycling Sequence in the C-tail of FPR2/ALX Receptor

PubMed Central

Thompson, Dawn; McArthur, Simon; Hislop, James N.; Flower, Roderick J.; Perretti, Mauro

2014-01-01

Formyl-peptide receptor type 2 (FPR2; also called ALX because it is the receptor for lipoxin A4) sustains a variety of biological responses relevant to the development and control of inflammation, yet the cellular regulation of this G-protein-coupled receptor remains unexplored. Here we report that, in response to peptide agonist activation, FPR2/ALX undergoes β-arrestin-mediated endocytosis followed by rapid recycling to the plasma membrane. We identify a transplantable recycling sequence that is both necessary and sufficient for efficient receptor recycling. Furthermore, removal of this C-terminal recycling sequence alters the endocytic fate of FPR2/ALX and evokes pro-apoptotic effects in response to agonist activation. This study demonstrates the importance of endocytic recycling in the anti-apoptotic properties of FPR2/ALX and identifies the molecular determinant required for modulation of this process fundamental for the control of inflammation. PMID:25326384

Pervasive sequence patents cover the entire human genome.

PubMed

Rosenfeld, Jeffrey A; Mason, Christopher E

2013-01-01

The scope and eligibility of patents for genetic sequences have been debated for decades, but a critical case regarding gene patents (Association of Molecular Pathologists v. Myriad Genetics) is now reaching the US Supreme Court. Recent court rulings have supported the assertion that such patents can provide intellectual property rights on sequences as small as 15 nucleotides (15mers), but an analysis of all current US patent claims and the human genome presented here shows that 15mer sequences from all human genes match at least one other gene. The average gene matches 364 other genes as 15mers; the breast-cancer-associated gene BRCA1 has 15mers matching at least 689 other genes. Longer sequences (1,000 bp) still showed extensive cross-gene matches. Furthermore, 15mer-length claims from bovine and other animal patents could also claim as much as 84% of the genes in the human genome. In addition, when we expanded our analysis to full-length patent claims on DNA from all US patents to date, we found that 41% of the genes in the human genome have been claimed. Thus, current patents for both short and long nucleotide sequences are extraordinarily non-specific and create an uncertain, problematic liability for genomic medicine, especially in regard to targeted re-sequencing and other sequence diagnostic assays.

A Molecular Method for the Identification of Honey Bee Subspecies Used by Beekeepers in Russia

PubMed Central

Syromyatnikov, Mikhail Y.; Borodachev, Anatoly V.; Kokina, Anastasia V.; Popov, Vasily N.

2018-01-01

Apis mellifera L. includes several recognized subspecies that differ in their biological properties and agricultural characteristics. Distinguishing between honey bee subspecies is complicated. We analyzed the Folmer region of the COX1 gene in honey bee subspecies cultivated at bee farms in Russia and identified subspecies-specific SNPs. DNA analysis revealed two clearly distinct haplogroups in A. mellifera mellifera. The first one was characterized by multiple cytosine-thymine (thymine–cytosine) transitions, one adenine-guanine substitution, and one thymine–adenine substitution. The nucleotide sequence of the second haplogroup coincided with sequences from other subspecies, except the unique C/A SNP at position 421 of the 658-bp Folmer region. A. mellifera carnica and A. mellifera carpatica could be distinguished from A. mellifera mellifera and A. mellifera caucasica by the presence of the A/G SNP at position 99 of the 658-bp Folmer region. The G/A SNP at position 448 was typical for A. mellifera carnica. A. mellifera caucasica COX1 sequence lacked all the above-mentioned sites. We developed a procedure for rapid identification of honey bee subspecies by PCR with restriction fragment length polymorphism (RFLP) using mutagenic primers. The developed molecular method for honey bee subspecies identification is fast and inexpensive. PMID:29382048

Complex folding and misfolding effects of deer-specific amino acid substitutions in the β2-α2 loop of murine prion protein

NASA Astrophysics Data System (ADS)

Agarwal, Sonya; Döring, Kristina; Gierusz, Leszek A.; Iyer, Pooja; Lane, Fiona M.; Graham, James F.; Goldmann, Wilfred; Pinheiro, Teresa J. T.; Gill, Andrew C.

2015-10-01

The β2-α2 loop of PrPC is a key modulator of disease-associated prion protein misfolding. Amino acids that differentiate mouse (Ser169, Asn173) and deer (Asn169, Thr173) PrPC appear to confer dramatically different structural properties in this region and it has been suggested that amino acid sequences associated with structural rigidity of the loop also confer susceptibility to prion disease. Using mouse recombinant PrP, we show that mutating residue 173 from Asn to Thr alters protein stability and misfolding only subtly, whilst changing Ser to Asn at codon 169 causes instability in the protein, promotes oligomer formation and dramatically potentiates fibril formation. The doubly mutated protein exhibits more complex folding and misfolding behaviour than either single mutant, suggestive of differential effects of the β2-α2 loop sequence on both protein stability and on specific misfolding pathways. Molecular dynamics simulation of protein structure suggests a key role for the solvent accessibility of Tyr168 in promoting molecular interactions that may lead to prion protein misfolding. Thus, we conclude that ‘rigidity’ in the β2-α2 loop region of the normal conformer of PrP has less effect on misfolding than other sequence-related effects in this region.

Targeted Re-Sequencing Emulsion PCR Panel for Myopathies: Results in 94 Cases.

PubMed

Punetha, Jaya; Kesari, Akanchha; Uapinyoying, Prech; Giri, Mamta; Clarke, Nigel F; Waddell, Leigh B; North, Kathryn N; Ghaoui, Roula; O'Grady, Gina L; Oates, Emily C; Sandaradura, Sarah A; Bönnemann, Carsten G; Donkervoort, Sandra; Plotz, Paul H; Smith, Edward C; Tesi-Rocha, Carolina; Bertorini, Tulio E; Tarnopolsky, Mark A; Reitter, Bernd; Hausmanowa-Petrusewicz, Irena; Hoffman, Eric P

2016-05-27

Molecular diagnostics in the genetic myopathies often requires testing of the largest and most complex transcript units in the human genome (DMD, TTN, NEB). Iteratively targeting single genes for sequencing has traditionally entailed high costs and long turnaround times. Exome sequencing has begun to supplant single targeted genes, but there are concerns regarding coverage and needed depth of the very large and complex genes that frequently cause myopathies. To evaluate efficiency of next-generation sequencing technologies to provide molecular diagnostics for patients with previously undiagnosed myopathies. We tested a targeted re-sequencing approach, using a 45 gene emulsion PCR myopathy panel, with subsequent sequencing on the Illumina platform in 94 undiagnosed patients. We compared the targeted re-sequencing approach to exome sequencing for 10 of these patients studied. We detected likely pathogenic mutations in 33 out of 94 patients with a molecular diagnostic rate of approximately 35%. The remaining patients showed variants of unknown significance (35/94 patients) or no mutations detected in the 45 genes tested (26/94 patients). Mutation detection rates for targeted re-sequencing vs. whole exome were similar in both methods; however exome sequencing showed better distribution of reads and fewer exon dropouts. Given that costs of highly parallel re-sequencing and whole exome sequencing are similar, and that exome sequencing now takes considerably less laboratory processing time than targeted re-sequencing, we recommend exome sequencing as the standard approach for molecular diagnostics of myopathies.

A Computational and Theoretical Study of Conductance in Hydrogen-bonded Molecular Junctions

NASA Astrophysics Data System (ADS)

Wimmer, Michael

This thesis is devoted to the theoretical and computational study of electron transport in molecular junctions where one or more hydrogen bonds are involved in the process. While electron transport through covalent bonds has been extensively studied, in recent work the focus has been shifted towards hydrogen-bonded systems due to their ubiquitous presence in biological systems and their potential in forming nano-junctions between molecular electronic devices and biological systems. This analysis allows us to significantly expand our comprehension of the experimentally observed result that the inclusion of hydrogen bonding in a molecular junction significantly impacts its transport properties, a fact that has important implications for our understanding of transport through DNA, and nano-biological interfaces in general. In part of this work I have explored the implications of quasiresonant transport in short chains of weakly-bonded molecular junctions involving hydrogen bonds. I used theoretical and computational analysis to interpret recent experiments and explain the role of Fano resonances in the transmission properties of the junction. In a different direction, I have undertaken the study of the transversal conduction through nucleotide chains that involve a variable number of different hydrogen bonds, e.g. NH˙˙˙O, OH˙˙˙O, and NH˙˙˙N, which are the three most prevalent hydrogen bonds in biological systems and organic electronics. My effort here has focused on the analysis of electronic descriptors that allow a simplified conceptual and computational understanding of transport properties. Specifically, I have expanded our previous work where the molecular polarizability was used as a conductance descriptor to include the possibility of atomic and bond partitions of the molecular polarizability. This is important because it affords an alternative molecular description of conductance that is not based on the conventional view of molecular orbitals as transport channels. My findings suggest that the hydrogen-bond networks are crucial in understanding the conductance of these junctions. A broader impact of this work pertains the fact that characterizing transport through hydrogen bonding networks may help in developing faster and cost-effective approaches to personalized medicine, to advance DNA sequencing and implantable electronics, and to progress in the design and application of new drugs.

In-Silico Analysis of Binding Site Features and Substrate Selectivity in Plant Flavonoid-3-O Glycosyltransferases (F3GT) through Molecular Modeling, Docking and Dynamics Simulation Studies

PubMed Central

Sharma, Ranu; Panigrahi, Priyabrata; Suresh, C.G.

2014-01-01

Flavonoids are a class of plant secondary metabolites that act as storage molecules, chemical messengers, as well as participate in homeostasis and defense processes. They possess pharmaceutical properties important for cancer treatment such as antioxidant and anti-tumor activities. The drug-related properties of flavonoids can be improved by glycosylation. The enzymes glycosyltransferases (GTs) glycosylate acceptor molecules in a regiospecific manner with the help of nucleotide sugar donor molecules. Several plant GTs have been characterized and their amino acid sequences determined. However, three-dimensional structures of only a few are reported. Here, phylogenetic analysis using amino acid sequences have identified a group of GTs with the same regiospecific activity. The structures of these closely related GTs were modeled using homologous GT structures. Their substrate binding sites were elaborated by docking flavonoid acceptor and UDP-sugar donor molecules in the modeled structures. Eight regions near the acceptor binding site in the N- and C- terminal domain of GTs have been identified that bind and specifically glycosylate the 3-OH group of acceptor flavonoids. Similarly, a conserved motif in the C-terminal domain is known to bind a sugar donor substrate. In certain GTs, the substitution of a specific glutamine by histidine in this domain changes the preference of sugar from glucose to galactose as a result of changed pattern of interactions. The molecular modeling, docking, and molecular dynamics simulation studies have revealed the chemical and topological features of the binding site and thus provided insights into the basis of acceptor and donor recognition by GTs. PMID:24667893

Morphological and molecular characterization of Eurytrema cladorchis parasitizing cattle (Bos indicus) in Bangladesh.

PubMed

Mohanta, Uday Kumar; Ichikawa-Seki, Madoka; Hayashi, Kei; Itagaki, Tadashi

2015-06-01

There is always controversy regarding identification of different species in the genus Eurytrema. Identification has been based mainly on morphology, which can be misleading and subject to differing interpretation among the scientists. Therefore, the aim of this study was to identify Eurytrema flukes both by morphology and molecular properties on the basis of 18-subunit ribosomal RNA (18S rRNA) gene as well as internal transcribed spacer 2 (ITS2) to clarify their phylogenetic status. Among six different agroecological areas of Bangladesh, 22 Eurytrema flukes were recovered from the bile ducts of 22 cattle in Bandarban, a hill district. The flukes were identified as Eurytrema cladorchis through morphometric and morphological studies. Phylogenetic analyses were conducted by neighbor-joining phylogram inferred from both 18S rRNA (1784 bp) gene and ITS2 (229 bp) sequences. A monophyletic clade was constructed by the E. cladorchis from Bangladesh; however, the clade was distinct from those formed by Eurytrema pancreaticum and Eurytrema coelomaticum. This study first described the existence of E. cladorchis from Bangladesh and may provide useful information for both morphological and molecular properties that may further help to clarify phylogenetic relationships within the genus Eurytrema and also for other digeneans.

Influenza virus sequence feature variant type analysis: evidence of a role for NS1 in influenza virus host range restriction.

PubMed

Noronha, Jyothi M; Liu, Mengya; Squires, R Burke; Pickett, Brett E; Hale, Benjamin G; Air, Gillian M; Galloway, Summer E; Takimoto, Toru; Schmolke, Mirco; Hunt, Victoria; Klem, Edward; García-Sastre, Adolfo; McGee, Monnie; Scheuermann, Richard H

2012-05-01

Genetic drift of influenza virus genomic sequences occurs through the combined effects of sequence alterations introduced by a low-fidelity polymerase and the varying selective pressures experienced as the virus migrates through different host environments. While traditional phylogenetic analysis is useful in tracking the evolutionary heritage of these viruses, the specific genetic determinants that dictate important phenotypic characteristics are often difficult to discern within the complex genetic background arising through evolution. Here we describe a novel influenza virus sequence feature variant type (Flu-SFVT) approach, made available through the public Influenza Research Database resource (www.fludb.org), in which variant types (VTs) identified in defined influenza virus protein sequence features (SFs) are used for genotype-phenotype association studies. Since SFs have been defined for all influenza virus proteins based on known structural, functional, and immune epitope recognition properties, the Flu-SFVT approach allows the rapid identification of the molecular genetic determinants of important influenza virus characteristics and their connection to underlying biological functions. We demonstrate the use of the SFVT approach to obtain statistical evidence for effects of NS1 protein sequence variations in dictating influenza virus host range restriction.

A comparative study of cold- and warm-adapted Endonucleases A using sequence analyses and molecular dynamics simulations.

PubMed

Michetti, Davide; Brandsdal, Bjørn Olav; Bon, Davide; Isaksen, Geir Villy; Tiberti, Matteo; Papaleo, Elena

2017-01-01

The psychrophilic and mesophilic endonucleases A (EndA) from Aliivibrio salmonicida (VsEndA) and Vibrio cholera (VcEndA) have been studied experimentally in terms of the biophysical properties related to thermal adaptation. The analyses of their static X-ray structures was no sufficient to rationalize the determinants of their adaptive traits at the molecular level. Thus, we used Molecular Dynamics (MD) simulations to compare the two proteins and unveil their structural and dynamical differences. Our simulations did not show a substantial increase in flexibility in the cold-adapted variant on the nanosecond time scale. The only exception is a more rigid C-terminal region in VcEndA, which is ascribable to a cluster of electrostatic interactions and hydrogen bonds, as also supported by MD simulations of the VsEndA mutant variant where the cluster of interactions was introduced. Moreover, we identified three additional amino acidic substitutions through multiple sequence alignment and the analyses of MD-based protein structure networks. In particular, T120V occurs in the proximity of the catalytic residue H80 and alters the interaction with the residue Y43, which belongs to the second coordination sphere of the Mg2+ ion. This makes T120V an amenable candidate for future experimental mutagenesis.

Molecular typing of Sporothrix schenckii isolates from cats in Malaysia.

PubMed

Kano, Rui; Okubo, Miki; Siew, Han Hock; Kamata, Hiroshi; Hasegawa, Atsuhiko

2015-04-01

Epidemiological data on the aetiologic agents of feline sporotrichosis in Malaysia have not been reported, though human sporotrichosis in Malaysia is reported to be transmitted primarily via cat scratch. To the best of our knowledge, the present report is the first study of the molecular epidemiology of Sporothrix schenckii isolates from cats with sporotrichosis in Malaysia. In the present work, we characterised 18 clinical isolates from cats in Malaysia based on molecular properties, including sequence analyses of the calmodulin gene and the rDNA ITS region and selective PCR of mating type (MAT) loci. In this study, isolates from feline sporotrichosis were identified as a S. schenckii sensu stricto by sequence analyses of the calmodulin gene and the internal transcribed spacer (ITS) region. Notably, phylogenetic analysis of the ITS confirmed assignment to clinical clade D (and not C) of S. schenckii sensu stricto. Therefore, clinical clade D of S. schenckii sensu stricto appeared to be the prevailing source of feline sporotrichosis in Malaysia. The ratio of MAT1-1-1:MAT1-2-1 in these Malaysian isolates was found to be 1 : 0. This result suggested that a clonal strain of S. schenckii is the prevailing causative agent of feline sporotrichosis in Malaysia. © 2015 Blackwell Verlag GmbH.

Conservation of a pH-sensitive structure in the C-terminal region of spider silk extends across the entire silk gene family.

PubMed

Strickland, Michelle; Tudorica, Victor; Řezáč, Milan; Thomas, Neil R; Goodacre, Sara L

2018-06-01

Spiders produce multiple silks with different physical properties that allow them to occupy a diverse range of ecological niches, including the underwater environment. Despite this functional diversity, past molecular analyses show a high degree of amino acid sequence similarity between C-terminal regions of silk genes that appear to be independent of the physical properties of the resulting silks; instead, this domain is crucial to the formation of silk fibers. Here, we present an analysis of the C-terminal domain of all known types of spider silk and include silk sequences from the spider Argyroneta aquatica, which spins the majority of its silk underwater. Our work indicates that spiders have retained a highly conserved mechanism of silk assembly, despite the extraordinary diversification of species, silk types and applications of silk over 350 million years. Sequence analysis of the silk C-terminal domain across the entire gene family shows the conservation of two uncommon amino acids that are implicated in the formation of a salt bridge, a functional bond essential to protein assembly. This conservation extends to the novel sequences isolated from A. aquatica. This finding is relevant to research regarding the artificial synthesis of spider silk, suggesting that synthesis of all silk types will be possible using a single process.

DNA mutation motifs in the genes associated with inherited diseases.

PubMed

Růžička, Michal; Kulhánek, Petr; Radová, Lenka; Čechová, Andrea; Špačková, Naďa; Fajkusová, Lenka; Réblová, Kamila

2017-01-01

Mutations in human genes can be responsible for inherited genetic disorders and cancer. Mutations can arise due to environmental factors or spontaneously. It has been shown that certain DNA sequences are more prone to mutate. These sites are termed hotspots and exhibit a higher mutation frequency than expected by chance. In contrast, DNA sequences with lower mutation frequencies than expected by chance are termed coldspots. Mutation hotspots are usually derived from a mutation spectrum, which reflects particular population where an effect of a common ancestor plays a role. To detect coldspots/hotspots unaffected by population bias, we analysed the presence of germline mutations obtained from HGMD database in the 5-nucleotide segments repeatedly occurring in genes associated with common inherited disorders, in particular, the PAH, LDLR, CFTR, F8, and F9 genes. Statistically significant sequences (mutational motifs) rarely associated with mutations (coldspots) and frequently associated with mutations (hotspots) exhibited characteristic sequence patterns, e.g. coldspots contained purine tract while hotspots showed alternating purine-pyrimidine bases, often with the presence of CpG dinucleotide. Using molecular dynamics simulations and free energy calculations, we analysed the global bending properties of two selected coldspots and two hotspots with a G/T mismatch. We observed that the coldspots were inherently more flexible than the hotspots. We assume that this property might be critical for effective mismatch repair as DNA with a mutation recognized by MutSα protein is noticeably bent.

Java web tools for PCR, in silico PCR, and oligonucleotide assembly and analysis.

PubMed

Kalendar, Ruslan; Lee, David; Schulman, Alan H

2011-08-01

The polymerase chain reaction is fundamental to molecular biology and is the most important practical molecular technique for the research laboratory. We have developed and tested efficient tools for PCR primer and probe design, which also predict oligonucleotide properties based on experimental studies of PCR efficiency. The tools provide comprehensive facilities for designing primers for most PCR applications and their combinations, including standard, multiplex, long-distance, inverse, real-time, unique, group-specific, bisulphite modification assays, Overlap-Extension PCR Multi-Fragment Assembly, as well as a programme to design oligonucleotide sets for long sequence assembly by ligase chain reaction. The in silico PCR primer or probe search includes comprehensive analyses of individual primers and primer pairs. It calculates the melting temperature for standard and degenerate oligonucleotides including LNA and other modifications, provides analyses for a set of primers with prediction of oligonucleotide properties, dimer and G-quadruplex detection, linguistic complexity, and provides a dilution and resuspension calculator. Copyright © 2011 Elsevier Inc. All rights reserved.

Bridging the physical scales in evolutionary biology: From protein sequence space to fitness of organisms and populations

PubMed Central

Bershtein, Shimon; Serohijos, Adrian W.R.; Shakhnovich, Eugene I.

2016-01-01

Bridging the gap between the molecular properties of proteins and organismal/population fitness is essential for understanding evolutionary processes. This task requires the integration of the several physical scales of biological organization, each defined by a distinct set of mechanisms and constraints, into a single unifying model. The molecular scale is dominated by the constraints imposed by the physico-chemical properties of proteins and their substrates, which give rise to trade-offs and epistatic (non-additive) effects of mutations. At the systems scale, biological networks modulate protein expression and can either buffer or enhance the fitness effects of mutations. The population scale is influenced by the mutational input, selection regimes, and stochastic changes affecting the size and structure of populations, which eventually determine the evolutionary fate of mutations. Here, we summarize the recent advances in theory, computer simulations, and experiments that advance our understanding of the links between various physical scales in biology. PMID:27810574

Bridging the physical scales in evolutionary biology: from protein sequence space to fitness of organisms and populations.

PubMed

Bershtein, Shimon; Serohijos, Adrian Wr; Shakhnovich, Eugene I

2017-02-01

Bridging the gap between the molecular properties of proteins and organismal/population fitness is essential for understanding evolutionary processes. This task requires the integration of the several physical scales of biological organization, each defined by a distinct set of mechanisms and constraints, into a single unifying model. The molecular scale is dominated by the constraints imposed by the physico-chemical properties of proteins and their substrates, which give rise to trade-offs and epistatic (non-additive) effects of mutations. At the systems scale, biological networks modulate protein expression and can either buffer or enhance the fitness effects of mutations. The population scale is influenced by the mutational input, selection regimes, and stochastic changes affecting the size and structure of populations, which eventually determine the evolutionary fate of mutations. Here, we summarize the recent advances in theory, computer simulations, and experiments that advance our understanding of the links between various physical scales in biology. Copyright © 2016 Elsevier Ltd. All rights reserved.

Copper metallothioneins.

PubMed

Calvo, Jenifer; Jung, Hunmin; Meloni, Gabriele

2017-04-01

Metallothioneins (MTs) are a class of low molecular weight and cysteine-rich metal binding proteins present in all the branches of the tree of life. MTs efficiently bind with high affinity several essential and toxic divalent and monovalent transition metals by forming characteristic polynuclear metal-thiolate clusters within their structure. MTs fulfil multiple biological functions related to their metal binding properties, with essential roles in both Zn(II) and Cu(I) homeostasis as well as metal detoxification. Depending on the organism considered, the primary sequence, and the specific physiological and metabolic status, Cu(I)-bound MT isoforms have been isolated, and their chemistry and biology characterized. Besides the recognized role in the biochemistry of divalent metals, it is becoming evident that unique biological functions in selectively controlling copper levels, its reactivity as well as copper-mediated biochemical processes have evolved in some members of the MT superfamily. Selected examples are reviewed to highlight the peculiar chemical properties and biological functions of copper MTs. © 2016 IUBMB Life, 69(4):236-245, 2017. © 2017 International Union of Biochemistry and Molecular Biology.

Molecular simulation and mathematical modelling of glass transition temperature depression induced by CO2 plasticization in Polysulfone membranes

NASA Astrophysics Data System (ADS)

Lock, S. S. M.; Lau, K. K.; Lock Sow Mei, Irene; Shariff, A. M.; Yeong, Y. F.; Bustam, A. M.

2017-08-01

A sequence of molecular modelling procedure has been proposed to simulate experimentally validated membrane structure characterizing the effect of CO2 plasticization, whereby it can be subsequently employed to elucidate the depression in glass transition temperature (Tg ). Based on the above motivation, unswollen and swollen Polysulfone membrane structures with different CO2 loadings have been constructed, whereby the accuracy has been validated through good compliance with experimentally measured physical properties. It is found that the presence of CO2 constitutes to enhancement in polymeric chain relaxation, which consequently promotes the enlargement of molecular spacing and causes dilation in the membrane matrix. A series of glass transition temperature treatment has been conducted on the verified molecular structure to elucidate the effect of CO2 loadings to the depression in Tg induced by plasticization. Subsequently, a modified Michealis-Menten (M-M) function has been implemented to quantify the effect of CO2 loading attributed to plasticization towards Tg .

Molecular characterization of the host defense activity of the barrier to autointegration factor against vaccinia virus.

PubMed

Ibrahim, Nouhou; Wicklund, April; Wiebe, Matthew S

2011-11-01

The barrier to autointegration factor (BAF) is an essential cellular protein with functions in mitotic nuclear reassembly, retroviral preintegration complex stability, and transcriptional regulation. Molecular properties of BAF include the ability to bind double-stranded DNA in a sequence-independent manner, homodimerize, and bind proteins containing a LEM domain. These capabilities allow BAF to compact DNA and assemble higher-order nucleoprotein complexes, the nature of which is poorly understood. Recently, it was revealed that BAF also acts as a potent host defense against poxviral DNA replication in the cytoplasm. Here, we extend these observations by examining the molecular mechanism through which BAF acts as a host defense against vaccinia virus replication and cytoplasmic DNA in general. Interestingly, BAF rapidly relocalizes to transfected DNA from a variety of sources, demonstrating that BAF's activity as a host defense factor is not limited to poxviral infection. BAF's relocalization to cytoplasmic foreign DNA is highly dependent upon its DNA binding and dimerization properties but does not appear to require its LEM domain binding activity. However, the LEM domain protein emerin is recruited to cytoplasmic DNA in a BAF-dependent manner during both transfection and vaccinia virus infection. Finally, we demonstrate that the DNA binding and dimerization capabilities of BAF are essential for its function as an antipoxviral effector, while the presence of emerin is not required. Together, these data provide further mechanistic insight into which of BAF's molecular properties are employed by cells to impair the replication of poxviruses or respond to foreign DNA in general.

Solid phase sequencing of biopolymers

DOEpatents

Cantor, Charles; Koster, Hubert

2010-09-28

This invention relates to methods for detecting and sequencing target nucleic acid sequences, to mass modified nucleic acid probes and arrays of probes useful in these methods, and to kits and systems which contain these probes. Useful methods involve hybridizing the nucleic acids or nucleic acids which represent complementary or homologous sequences of the target to an array of nucleic acid probes. These probes comprise a single-stranded portion, an optional double-stranded portion and a variable sequence within the single-stranded portion. The molecular weights of the hybridized nucleic acids of the set can be determined by mass spectroscopy, and the sequence of the target determined from the molecular weights of the fragments. Nucleic acids whose sequences can be determined include DNA or RNA in biological samples such as patient biopsies and environmental samples. Probes may be fixed to a solid support such as a hybridization chip to facilitate automated molecular weight analysis and identification of the target sequence.

Molecular characterization, phylogeny analysis and pathogenicity of a Muscovy duck adenovirus strain isolated in China in 2014

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

Zhang, Xinheng; Key Laboratory of Animal Health Aquaculture and Environmental Control, Guangzhou, Guangdong 510642; South China Collaborative Innovation Center for Poultry Disease Control and Product Safety, Guangzhou 510642

This study aimed to characterize a novel adenovirus (AdV) isolated from diseased Muscovy ducks in China. After the AdV was successfully propagated in duck embryo fibroblasts, the morphological and physicochemical properties of the virions were studied by electron microscopy and different tests. The results of the analyses were in conformity with AdV properties. The full genome sequence was determined and analyzed. The new isolate (named CH-GD-12-2014) shared over 91% sequence identity with duck AdV-2 representing the species Duck aviadenovirus B. The most important distinguishing feature between the two DAdV strains was the presence of a second fiber gene in themore » Chinese isolate. Phylogeny reconstruction confirmed the affiliation of the virus with goose and duck AdVs in the genus Aviadenovirus. Experimental infection resulted in embryo death, and intramuscular inoculation provoked morbidity and mortality among ducks and chickens. - Highlights: • A duck adenovirus type 3 was isolated and the complete genome of DAdV-3 was obtained. • Physicochemical properties and electron microscopy were researched. • Pathogenicity of duck adenovirus type 3 was researched.« less

Growth and Properties of Lattice Matched GaAsSbN Epilayer on GaAs for Solar Cell Applications

NASA Technical Reports Server (NTRS)

Bharatan, Sudhakar; Iyer, Shanthi; Matney, Kevin; Collis, Ward J.; Nunna Kalyan; Li, Jia; Wu, Liangjin; McGuire, Kristopher; McNeil, Laurie E.

2006-01-01

The growth and properties of GaAsSbN single quantum wells (SQWs) are investigated in this work. The heterostructures were grown on GaAs substrates in an elemental solid source molecular beam epitaxy (MBE) system assisted with a RF plasma nitrogen source. A systematic study has been carried out to determine the influence of various growth conditions, such as the growth temperature and the source shutter-opening sequence, on the quality of the grown layers and the incorporation of N and Sb. The effects of ex situ and in situ annealing under As overpressure on the optical properties of the layers have also been investigated. Substrate temperature in the range of 450-470 C was found to be optimum. Simultaneous opening of the source shutters was found to yield sharper QW interfaces. N and Sb incorporations were found to depend strongly upon substrate temperatures and source shutter opening sequences. A significant increase in PL intensity with a narrowing of PL line shape and blue shift in emission energy were observed on annealing the GaAsSbN/GaAs SQW, with in situ annealing under As overpressure providing better results, compared to ex situ annealing.

Creating complex molecular topologies by configuring DNA four-way junctions

NASA Astrophysics Data System (ADS)

Liu, Di; Chen, Gang; Akhter, Usman; Cronin, Timothy M.; Weizmann, Yossi

2016-10-01

The realization of complex topologies at the molecular level represents a grand challenge in chemistry. This necessitates the manipulation of molecular interactions with high precision. Here we show that single-stranded DNA (ssDNA) knots and links can be created by utilizing the inherent topological properties that pertain to the DNA four-way junction, at which the two helical strands form a node and can be configured conveniently and connected for complex topological construction. Using this strategy, we produced series of ssDNA topoisomers with the same sequences. By finely designing the curvature and torsion, double-stranded DNA knots were accessed by hybridizing and ligating the complementary strands with the knotted ssDNA templates. Furthermore, we demonstrate the use of a constructed ssDNA knot both to probe the topological conversion catalysed by DNA topoisomerase and to study the DNA replication under topological constraint.

[Molecular aspects of allergy to plant products. Part II. Pathogenesis-related proteins (PRs), apple allergenicity governed by Mal d 1 gene].

PubMed

Bokszczanin, Kamila Ł; Przybyła, Andrzej A

2012-03-01

Of the plant allergens listed in the Official Allergen Database of the International Union of Immunological Societies, approximately 25% belong to the group of pathogenesis-related proteins (PRs). They have been classified into 17 PR families based on similarities in their amino acid sequence, enzymatic activities, or other functional properties. Plant-derived allergens have been identified with sequence similarities to PR families 2, 3, 4, 5, 8, 10, and 14. The main birch allergen in northern Europe is a class 10 (PR-10) protein from the European white birch (Betula pendula) termed Bet v 1. Pollen of other Fagales species contains PR-10 homologues that share epitopes with Bet v 1, as do several fruits, nuts and vegetables. Among the plant food fruits of the Rosaceae family are the most frequently responsible for allergenic reactions. It is documented, that approximately 2% of European population is allergic to apples. The article presents molecular characterization of PR-10 proteins with regard to their structure and function as well as apple Mal d 1 gene-determined allergenicity.

The Function of Embryonic Stem Cell-expressed RAS (E-RAS), a Unique RAS Family Member, Correlates with Its Additional Motifs and Its Structural Properties.

PubMed

Nakhaei-Rad, Saeideh; Nakhaeizadeh, Hossein; Kordes, Claus; Cirstea, Ion C; Schmick, Malte; Dvorsky, Radovan; Bastiaens, Philippe I H; Häussinger, Dieter; Ahmadian, Mohammad Reza

2015-06-19

E-RAS is a member of the RAS family specifically expressed in embryonic stem cells, gastric tumors, and hepatic stellate cells. Unlike classical RAS isoforms (H-, N-, and K-RAS4B), E-RAS has, in addition to striking and remarkable sequence deviations, an extended 38-amino acid-long unique N-terminal region with still unknown functions. We investigated the molecular mechanism of E-RAS regulation and function with respect to its sequence and structural features. We found that N-terminal extension of E-RAS is important for E-RAS signaling activity. E-RAS protein most remarkably revealed a different mode of effector interaction as compared with H-RAS, which correlates with deviations in the effector-binding site of E-RAS. Of all these residues, tryptophan 79 (arginine 41 in H-RAS), in the interswitch region, modulates the effector selectivity of RAS proteins from H-RAS to E-RAS features. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

FUV Spectroscopy Of Outflows And Disks Around The Intermediate Mass Pre-main-sequence Stars HD135344B And HD104237

NASA Astrophysics Data System (ADS)

Brown, Alexander; Herczeg, G.; Brown, J. M.; Walter, F. M.; Ayres, T. R.; DAOof TAU Team

2011-01-01

The intermediate-mass, pre-main-sequence (Herbig Ae/Fe) stars HD135344B (F4) and HD104237 (A8 IV-V) are both still surrounded by almost face-on circumstellar disks. The disk around HD135344B is a ``transitional'' disk with a 25 AU radius cleared inner hole but still with some gas and dust very close to the star. We have obtained FUV spectra of these stars using the HST COS and STIS spectrographs that show that both stars have dramatic high-velocity (terminal velocity = 300-400 km/s) outflows and rich fluorescently-excited molecular hydrogen emission, originating primarily from warm gas in their disks. We present these FUV spectra and outline the outflow and disk properties implied by the observed emission and absorption line profiles. The profiles and widths of the molecular hydrogen lines provide strong constraints on the location of the emitting regions. This work is supported by HST grants for GO projects 11828 and 11616, and Chandra grant GO9-0015X to the University of Colorado.

The opportunities and challenges of large-scale molecular approaches to songbird neurobiology

PubMed Central

Mello, C.V.; Clayton, D.F.

2014-01-01

High-through put methods for analyzing genome structure and function are having a large impact in song-bird neurobiology. Methods include genome sequencing and annotation, comparative genomics, DNA microarrays and transcriptomics, and the development of a brain atlas of gene expression. Key emerging findings include the identification of complex transcriptional programs active during singing, the robust brain expression of non-coding RNAs, evidence of profound variations in gene expression across brain regions, and the identification of molecular specializations within song production and learning circuits. Current challenges include the statistical analysis of large datasets, effective genome curations, the efficient localization of gene expression changes to specific neuronal circuits and cells, and the dissection of behavioral and environmental factors that influence brain gene expression. The field requires efficient methods for comparisons with organisms like chicken, which offer important anatomical, functional and behavioral contrasts. As sequencing costs plummet, opportunities emerge for comparative approaches that may help reveal evolutionary transitions contributing to vocal learning, social behavior and other properties that make songbirds such compelling research subjects. PMID:25280907

Application of next-generation sequencing for rapid marker development in molecular plant breeding: a case study on anthracnose disease resistance in Lupinus angustifolius L.

PubMed Central

2012-01-01

Background In the last 30 years, a number of DNA fingerprinting methods such as RFLP, RAPD, AFLP, SSR, DArT, have been extensively used in marker development for molecular plant breeding. However, it remains a daunting task to identify highly polymorphic and closely linked molecular markers for a target trait for molecular marker-assisted selection. The next-generation sequencing (NGS) technology is far more powerful than any existing generic DNA fingerprinting methods in generating DNA markers. In this study, we employed a grain legume crop Lupinus angustifolius (lupin) as a test case, and examined the utility of an NGS-based method of RAD (restriction-site associated DNA) sequencing as DNA fingerprinting for rapid, cost-effective marker development tagging a disease resistance gene for molecular breeding. Results Twenty informative plants from a cross of RxS (disease resistant x susceptible) in lupin were subjected to RAD single-end sequencing by multiplex identifiers. The entire RAD sequencing products were resolved in two lanes of the 16-lanes per run sequencing platform Solexa HiSeq2000. A total of 185 million raw reads, approximately 17 Gb of sequencing data, were collected. Sequence comparison among the 20 test plants discovered 8207 SNP markers. Filtration of DNA sequencing data with marker identification parameters resulted in the discovery of 38 molecular markers linked to the disease resistance gene Lanr1. Five randomly selected markers were converted into cost-effective, simple PCR-based markers. Linkage analysis using marker genotyping data and disease resistance phenotyping data on a F8 population consisting of 186 individual plants confirmed that all these five markers were linked to the R gene. Two of these newly developed sequence-specific PCR markers, AnSeq3 and AnSeq4, flanked the target R gene at a genetic distance of 0.9 centiMorgan (cM), and are now replacing the markers previously developed by a traditional DNA fingerprinting method for marker-assisted selection in the Australian national lupin breeding program. Conclusions We demonstrated that more than 30 molecular markers linked to a target gene of agronomic trait of interest can be identified from a small portion (1/8) of one sequencing run on HiSeq2000 by applying NGS based RAD sequencing in marker development. The markers developed by the strategy described in this study are all co-dominant SNP markers, which can readily be converted into high throughput multiplex format or low-cost, simple PCR-based markers desirable for large scale marker implementation in plant breeding programs. The high density and closely linked molecular markers associated with a target trait help to overcome a major bottleneck for implementation of molecular markers on a wide range of germplasm in breeding programs. We conclude that application of NGS based RAD sequencing as DNA fingerprinting is a very rapid and cost-effective strategy for marker development in molecular plant breeding. The strategy does not require any prior genome knowledge or molecular information for the species under investigation, and it is applicable to other plant species. PMID:22805587

Application of next-generation sequencing for rapid marker development in molecular plant breeding: a case study on anthracnose disease resistance in Lupinus angustifolius L.

PubMed

Yang, Huaan; Tao, Ye; Zheng, Zequn; Li, Chengdao; Sweetingham, Mark W; Howieson, John G

2012-07-17

In the last 30 years, a number of DNA fingerprinting methods such as RFLP, RAPD, AFLP, SSR, DArT, have been extensively used in marker development for molecular plant breeding. However, it remains a daunting task to identify highly polymorphic and closely linked molecular markers for a target trait for molecular marker-assisted selection. The next-generation sequencing (NGS) technology is far more powerful than any existing generic DNA fingerprinting methods in generating DNA markers. In this study, we employed a grain legume crop Lupinus angustifolius (lupin) as a test case, and examined the utility of an NGS-based method of RAD (restriction-site associated DNA) sequencing as DNA fingerprinting for rapid, cost-effective marker development tagging a disease resistance gene for molecular breeding. Twenty informative plants from a cross of RxS (disease resistant x susceptible) in lupin were subjected to RAD single-end sequencing by multiplex identifiers. The entire RAD sequencing products were resolved in two lanes of the 16-lanes per run sequencing platform Solexa HiSeq2000. A total of 185 million raw reads, approximately 17 Gb of sequencing data, were collected. Sequence comparison among the 20 test plants discovered 8207 SNP markers. Filtration of DNA sequencing data with marker identification parameters resulted in the discovery of 38 molecular markers linked to the disease resistance gene Lanr1. Five randomly selected markers were converted into cost-effective, simple PCR-based markers. Linkage analysis using marker genotyping data and disease resistance phenotyping data on a F8 population consisting of 186 individual plants confirmed that all these five markers were linked to the R gene. Two of these newly developed sequence-specific PCR markers, AnSeq3 and AnSeq4, flanked the target R gene at a genetic distance of 0.9 centiMorgan (cM), and are now replacing the markers previously developed by a traditional DNA fingerprinting method for marker-assisted selection in the Australian national lupin breeding program. We demonstrated that more than 30 molecular markers linked to a target gene of agronomic trait of interest can be identified from a small portion (1/8) of one sequencing run on HiSeq2000 by applying NGS based RAD sequencing in marker development. The markers developed by the strategy described in this study are all co-dominant SNP markers, which can readily be converted into high throughput multiplex format or low-cost, simple PCR-based markers desirable for large scale marker implementation in plant breeding programs. The high density and closely linked molecular markers associated with a target trait help to overcome a major bottleneck for implementation of molecular markers on a wide range of germplasm in breeding programs. We conclude that application of NGS based RAD sequencing as DNA fingerprinting is a very rapid and cost-effective strategy for marker development in molecular plant breeding. The strategy does not require any prior genome knowledge or molecular information for the species under investigation, and it is applicable to other plant species.

Molecular differences between deuterated and protonated polystyrenes using reversed-phase high-performance liquid chromatography.

PubMed

Kayillo, Sindy; Gray, Michael J; Shalliker, R Andrew; Dennis, Gary R

2005-05-06

Isotopic substitution is a technique used to highlight particular bonds within a molecule for kinetic, spectroscopic and structure analysis. It is presumed that although some properties such as stretching frequencies will not be the same for substituted analogues, the chemical interactions will not vary appreciably as a function of labelling. Reversed-phase liquid chromatography has been used to demonstrate that there are significant differences between the chromatographic behaviour of a sequence of deuterated and protonated oligomeric polystyrenes. Two-dimensional reversed-phase liquid chromatography was used to show that even the diasteromers of the oligomers (n = 5) have retention mechanisms that are dependent on the subtle changes to the molecular conformation and electronic structure, which are a consequence of deuteration.

New molecular features of cowpea bean (Vigna unguiculata, l. Walp) β-vignin.

PubMed

de Souza Ferreira, Ederlan; Capraro, Jessica; Sessa, Fabio; Magni, Chiara; Demonte, Aureluce; Consonni, Alessandro; Augusto Neves, Valdir; Maffud Cilli, Eduardo; Duranti, Marcello; Scarafoni, Alessio

2018-02-01

Cowpea seed β-vignin, a vicilin-like globulin, proved to exert various health favourable effects, including blood cholesterol reduction in animal models. The need of a simple scalable enrichment procedure for further studies for tailored applications of this seed protein is crucial. A chromatography-independent fractionation method allowing to obtain a protein preparation with a high degree of homogeneity was used. Further purification was pursued to deep the molecular characterisation of β-vignin. The results showed: (i) differing glycosylation patterns of the two constituent polypeptides, in agreement with amino acid sequence features; (ii) the seed accumulation of a gene product never identified before; (iii) metal binding capacity of native protein, a property observed only in few other legume seed vicilins.

Tracking and Motion Analysis of Crack Propagations in Crystals for Molecular Dynamics

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

Tsap, L V; Duchaineau, M; Goldgof, D B

2001-05-14

This paper presents a quantitative analysis for a discovery in molecular dynamics. Recent simulations have shown that velocities of crack propagations in crystals under certain conditions can become supersonic, which is contrary to classical physics. In this research, they present a framework for tracking and motion analysis of crack propagations in crystals. It includes line segment extraction based on Canny edge maps, feature selection based on physical properties, and subsequent tracking of primary and secondary wavefronts. This tracking is completely automated; it runs in real time on three 834-image sequences using forty 250 MHZ processors. Results supporting physical observations aremore » presented in terms of both feature tracking and velocity analysis.« less

SequenceCEROSENE: a computational method and web server to visualize spatial residue neighborhoods at the sequence level.

PubMed

Heinke, Florian; Bittrich, Sebastian; Kaiser, Florian; Labudde, Dirk

2016-01-01

To understand the molecular function of biopolymers, studying their structural characteristics is of central importance. Graphics programs are often utilized to conceive these properties, but with the increasing number of available structures in databases or structure models produced by automated modeling frameworks this process requires assistance from tools that allow automated structure visualization. In this paper a web server and its underlying method for generating graphical sequence representations of molecular structures is presented. The method, called SequenceCEROSENE (color encoding of residues obtained by spatial neighborhood embedding), retrieves the sequence of each amino acid or nucleotide chain in a given structure and produces a color coding for each residue based on three-dimensional structure information. From this, color-highlighted sequences are obtained, where residue coloring represent three-dimensional residue locations in the structure. This color encoding thus provides a one-dimensional representation, from which spatial interactions, proximity and relations between residues or entire chains can be deduced quickly and solely from color similarity. Furthermore, additional heteroatoms and chemical compounds bound to the structure, like ligands or coenzymes, are processed and reported as well. To provide free access to SequenceCEROSENE, a web server has been implemented that allows generating color codings for structures deposited in the Protein Data Bank or structure models uploaded by the user. Besides retrieving visualizations in popular graphic formats, underlying raw data can be downloaded as well. In addition, the server provides user interactivity with generated visualizations and the three-dimensional structure in question. Color encoded sequences generated by SequenceCEROSENE can aid to quickly perceive the general characteristics of a structure of interest (or entire sets of complexes), thus supporting the researcher in the initial phase of structure-based studies. In this respect, the web server can be a valuable tool, as users are allowed to process multiple structures, quickly switch between results, and interact with generated visualizations in an intuitive manner. The SequenceCEROSENE web server is available at https://biosciences.hs-mittweida.de/seqcerosene.

Computational Analysis of Molecular Interaction Networks Underlying Change of HIV-1 Resistance to Selected Reverse Transcriptase Inhibitors

PubMed Central

Kierczak, Marcin; Dramiński, Michał; Koronacki, Jacek; Komorowski, Jan

2010-01-01

Motivation Despite more than two decades of research, HIV resistance to drugs remains a serious obstacle in developing efficient AIDS treatments. Several computational methods have been developed to predict resistance level from the sequence of viral proteins such as reverse transcriptase (RT) or protease. These methods, while powerful and accurate, give very little insight into the molecular interactions that underly acquisition of drug resistance/hypersusceptibility. Here, we attempt at filling this gap by using our Monte Carlo feature selection and interdependency discovery method (MCFS-ID) to elucidate molecular interaction networks that characterize viral strains with altered drug resistance levels. Results We analyzed a number of HIV-1 RT sequences annotated with drug resistance level using the MCFS-ID method. This let us expound interdependency networks that characterize change of drug resistance to six selected RT inhibitors: Abacavir, Lamivudine, Stavudine, Zidovudine, Tenofovir and Nevirapine. The networks consider interdependencies at the level of physicochemical properties of mutating amino acids, eg,: polarity. We mapped each network on the 3D structure of RT in attempt to understand the molecular meaning of interacting pairs. The discovered interactions describe several known drug resistance mechanisms and, importantly, some previously unidentified ones. Our approach can be easily applied to a whole range of problems from the domain of protein engineering. Availability A portable Java implementation of our MCFS-ID method is freely available for academic users and can be obtained at: http://www.ipipan.eu/staff/m.draminski/software.htm. PMID:21234299

Prototypical Recombinant Multi-Protease-Inhibitor-Resistant Infectious Molecular Clones of Human Immunodeficiency Virus Type 1

PubMed Central

Varghese, Vici; Mitsuya, Yumi; Fessel, W. Jeffrey; Liu, Tommy F.; Melikian, George L.; Katzenstein, David A.; Schiffer, Celia A.; Holmes, Susan P.

2013-01-01

The many genetic manifestations of HIV-1 protease inhibitor (PI) resistance present challenges to research into the mechanisms of PI resistance and the assessment of new PIs. To address these challenges, we created a panel of recombinant multi-PI-resistant infectious molecular clones designed to represent the spectrum of clinically relevant multi-PI-resistant viruses. To assess the representativeness of this panel, we examined the sequences of the panel's viruses in the context of a correlation network of PI resistance amino acid substitutions in sequences from more than 10,000 patients. The panel of recombinant infectious molecular clones comprised 29 of 41 study-defined PI resistance amino acid substitutions and 23 of the 27 tightest amino acid substitution clusters. Based on their phenotypic properties, the clones were classified into four groups with increasing cross-resistance to the PIs most commonly used for salvage therapy: lopinavir (LPV), tipranavir (TPV), and darunavir (DRV). The panel of recombinant infectious molecular clones has been made available without restriction through the NIH AIDS Research and Reference Reagent Program. The public availability of the panel makes it possible to compare the inhibitory activities of different PIs with one another. The diversity of the panel and the high-level PI resistance of its clones suggest that investigational PIs active against the clones in this panel will retain antiviral activity against most if not all clinically relevant PI-resistant viruses. PMID:23796938

Computational Analysis of Molecular Interaction Networks Underlying Change of HIV-1 Resistance to Selected Reverse Transcriptase Inhibitors.

PubMed

Kierczak, Marcin; Dramiński, Michał; Koronacki, Jacek; Komorowski, Jan

2010-12-12

Despite more than two decades of research, HIV resistance to drugs remains a serious obstacle in developing efficient AIDS treatments. Several computational methods have been developed to predict resistance level from the sequence of viral proteins such as reverse transcriptase (RT) or protease. These methods, while powerful and accurate, give very little insight into the molecular interactions that underly acquisition of drug resistance/hypersusceptibility. Here, we attempt at filling this gap by using our Monte Carlo feature selection and interdependency discovery method (MCFS-ID) to elucidate molecular interaction networks that characterize viral strains with altered drug resistance levels. We analyzed a number of HIV-1 RT sequences annotated with drug resistance level using the MCFS-ID method. This let us expound interdependency networks that characterize change of drug resistance to six selected RT inhibitors: Abacavir, Lamivudine, Stavudine, Zidovudine, Tenofovir and Nevirapine. The networks consider interdependencies at the level of physicochemical properties of mutating amino acids, eg,: polarity. We mapped each network on the 3D structure of RT in attempt to understand the molecular meaning of interacting pairs. The discovered interactions describe several known drug resistance mechanisms and, importantly, some previously unidentified ones. Our approach can be easily applied to a whole range of problems from the domain of protein engineering. A portable Java implementation of our MCFS-ID method is freely available for academic users and can be obtained at: http://www.ipipan.eu/staff/m.draminski/software.htm.

Morphological and molecular characterization of a marine fish trypanosome from South Africa, including its development in a leech vector

PubMed Central

2014-01-01

Background Trypanosomes are ubiquitous blood parasites of marine and freshwater fishes, typically transmitted by aquatic leeches. Phylogenetic studies have been dominated by examples derived from freshwater fishes, with few marine representatives. Furthermore, life cycle studies on marine fish trypanosomes have focused on those of the northern hemisphere. In this investigation, we have examined the life cycle and molecular taxonomy of a marine fish trypanosome from South Africa. Methods To locate trypanosome stages, leeches were removed from fishes captured on the west and south coasts of South Africa, and fish blood films and leech squashes were Giemsa-stained and screened; leeches were also examined histologically. To determine whether trypanosome stages in fishes and leeches were of the same genotype, DNA was extracted from Giemsa-stained fish blood films and leech squashes, and from fish whole blood. Fragments of the 18S rRNA gene were amplified by PCR using trypanosome-specific primers and sequenced. Resulting sequence data were compared with each other and with published trypanosome 18S rDNA sequences, and used for phylogenetic analysis. Results Trypanosomes were detected in blood films from fishes of the families Clinidae, Blenniidae and Gobiidae. The flagellates ranged in size and staining properties within the films and across fish hosts. In squashes and histological sections of adult and juvenile leeches, identified as Zeylanicobdella arugamensis, trypanosome developmental stages were predominantly slender epimastigotes. Sequence data showed that trypanosomes derived from fishes were identical, irrespective of whether they were small or large forms; sequences derived largely from leech epimastigotes were also identical to those obtained from fish trypanosomes. Fish and leech trypanosome sequences fell into a marine fish aquatic clade, and aligned most closely with two trypanosome sequences from marine fishes off Norway. Conclusions Combined morphological and molecular methods indicate that the trypanosomes examined here represent a single pleomorphic species, rather than the three species described originally. This species is identified as Trypanosoma nudigobii Fantham, 1919 with the leech Z. arugamensis as its vector, and T. capigobii Fantham, 1919 and T. blenniclini Fantham, 1930 are regarded as junior synonyms of the species. Phylogenetic analysis establishes its affinity with marine fish trypanosomes off Norway. PMID:24460725

Nucleotide sequences of immunoglobulin eta genes of chimpanzee and orangutan: DNA molecular clock and hominoid evolution

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

Sakoyama, Y.; Hong, K.J.; Byun, S.M.

To determine the phylogenetic relationships among hominoids and the dates of their divergence, the complete nucleotide sequences of the constant region of the immunoglobulin eta-chain (C/sub eta1/) genes from chimpanzee and orangutan have been determined. These sequences were compared with the human eta-chain constant-region sequence. A molecular clock (silent molecular clock), measured by the degree of sequence divergence at the synonymous (silent) positions of protein-encoding regions, was introduced for the present study. From the comparison of nucleotide sequences of ..cap alpha../sub 1/-antitrypsin and ..beta..- and delta-globulin genes between humans and Old World monkeys, the silent molecular clock was calibrated: themore » mean evolutionary rate of silent substitution was determined to be 1.56 x 10/sup -9/ substitutions per site per year. Using the silent molecular clock, the mean divergence dates of chimpanzee and orangutan from the human lineage were estimated as 6.4 +/- 2.6 million years and 17.3 +/- 4.5 million years, respectively. It was also shown that the evolutionary rate of primate genes is considerably slower than those of other mammalian genes.« less

Sequence-controlled methacrylic multiblock copolymers via sulfur-free RAFT emulsion polymerization

NASA Astrophysics Data System (ADS)

Engelis, Nikolaos G.; Anastasaki, Athina; Nurumbetov, Gabit; Truong, Nghia P.; Nikolaou, Vasiliki; Shegiwal, Ataulla; Whittaker, Michael R.; Davis, Thomas P.; Haddleton, David M.

2017-02-01

Translating the precise monomer sequence control achieved in nature over macromolecular structure (for example, DNA) to whole synthetic systems has been limited due to the lack of efficient synthetic methodologies. So far, chemists have only been able to synthesize monomer sequence-controlled macromolecules by means of complex, time-consuming and iterative chemical strategies such as solid-state Merrifield-type approaches or molecularly dissolved solution-phase systems. Here, we report a rapid and quantitative synthesis of sequence-controlled multiblock polymers in discrete stable nanoscale compartments via an emulsion polymerization approach in which a vinyl-terminated macromolecule is used as an efficient chain-transfer agent. This approach is environmentally friendly, fully translatable to industry and thus represents a significant advance in the development of complex macromolecule synthesis, where a high level of molecular precision or monomer sequence control confers potential for molecular targeting, recognition and biocatalysis, as well as molecular information storage.

Variation in Orthologous Shell-Forming Proteins Contribute to Molluscan Shell Diversity

PubMed Central

Jackson, Daniel J.; Reim, Laurin; Randow, Clemens; Cerveau, Nicolas; Degnan, Bernard M.; Fleck, Claudia

2017-01-01

Abstract Despite the evolutionary success and ancient heritage of the molluscan shell, little is known about the molecular details of its formation, evolutionary origins, or the interactions between the material properties of the shell and its organic constituents. In contrast to this dearth of information, a growing collection of molluscan shell-forming proteomes and transcriptomes suggest they are comprised of both deeply conserved, and lineage specific elements. Analyses of these sequence data sets have suggested that mechanisms such as exon shuffling, gene co-option, and gene family expansion facilitated the rapid evolution of shell-forming proteomes and supported the diversification of this phylum specific structure. In order to further investigate and test these ideas we have examined the molecular features and spatial expression patterns of two shell-forming genes (Lustrin and ML1A2) and coupled these observations with materials properties measurements of shells from a group of closely related gastropods (abalone). We find that the prominent “GS” domain of Lustrin, a domain believed to confer elastomeric properties to the shell, varies significantly in length between the species we investigated. Furthermore, the spatial expression patterns of Lustrin and ML1A2 also vary significantly between species, suggesting that both protein architecture, and the regulation of spatial gene expression patterns, are important drivers of molluscan shell evolution. Variation in these molecular features might relate to certain materials properties of the shells of these species. These insights reveal an important and underappreciated source of variation within shell-forming proteomes that must contribute to the diversity of molluscan shell phenotypes. PMID:28961798

Sequencing consolidates molecular markers with plant breeding practice.

PubMed

Yang, Huaan; Li, Chengdao; Lam, Hon-Ming; Clements, Jonathan; Yan, Guijun; Zhao, Shancen

2015-05-01

Plenty of molecular markers have been developed by contemporary sequencing technologies, whereas few of them are successfully applied in breeding, thus we present a review on how sequencing can facilitate marker-assisted selection in plant breeding. The growing global population and shrinking arable land area require efficient plant breeding. Novel strategies assisted by certain markers have proven effective for genetic gains. Fortunately, cutting-edge sequencing technologies bring us a deluge of genomes and genetic variations, enlightening the potential of marker development. However, a large gap still exists between the potential of molecular markers and actual plant breeding practices. In this review, we discuss marker-assisted breeding from a historical perspective, describe the road from crop sequencing to breeding, and highlight how sequencing facilitates the application of markers in breeding practice.

Molecular Identification of Ectomycorrhizal Mycelium in Soil Horizons

PubMed Central

Landeweert, Renske; Leeflang, Paula; Kuyper, Thom W.; Hoffland, Ellis; Rosling, Anna; Wernars, Karel; Smit, Eric

2003-01-01

Molecular identification techniques based on total DNA extraction provide a unique tool for identification of mycelium in soil. Using molecular identification techniques, the ectomycorrhizal (EM) fungal community under coniferous vegetation was analyzed. Soil samples were taken at different depths from four horizons of a podzol profile. A basidiomycete-specific primer pair (ITS1F-ITS4B) was used to amplify fungal internal transcribed spacer (ITS) sequences from total DNA extracts of the soil horizons. Amplified basidiomycete DNA was cloned and sequenced, and a selection of the obtained clones was analyzed phylogenetically. Based on sequence similarity, the fungal clone sequences were sorted into 25 different fungal groups, or operational taxonomic units (OTUs). Out of 25 basidiomycete OTUs, 7 OTUs showed high nucleotide homology (≥99%) with known EM fungal sequences and 16 were found exclusively in the mineral soil. The taxonomic positions of six OTUs remained unclear. OTU sequences were compared to sequences from morphotyped EM root tips collected from the same sites. Of the 25 OTUs, 10 OTUs had ≥98% sequence similarity with these EM root tip sequences. The present study demonstrates the use of molecular techniques to identify EM hyphae in various soil types. This approach differs from the conventional method of EM root tip identification and provides a novel approach to examine EM fungal communities in soil. PMID:12514012

Peptide-Directed PdAu Nanoscale Surface Segregation: Toward Controlled Bimetallic Architecture for Catalytic Materials

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

Bedford, Nicholas M.; Showalter, Allison R.; Woehl, Taylor J.

Bimetallic nanoparticles are of immense scientific and technological interest given the synergistic properties observed when mixing two different metallic species at the nanoscale. This is particularly prevalent in catalysis, where bimetallic nanoparticles often exhibit improved catalytic activity and durability over their monometallic counterparts. Yet despite intense research efforts, little is understood regarding how to optimize bimetallic surface composition and structure synthetically using rational design principles. Recently, it has been demonstrated that peptide-enabled routes for nanoparticle synthesis result in materials with sequence-dependent catalytic properties, providing an opportunity for rational design through sequence manipulation. In this study, bimetallic PdAu nanoparticles are synthesizedmore » with a small set of peptides containing known Pd and Au binding motifs. The resulting nanoparticles were extensively characterized using high-resolution scanning transmission electron microscopy, X-ray absorption spectroscopy and high-energy X-ray diffraction coupled to atomic pair distribution function analysis. Structural information obtained from synchrotron radiation methods were then used to generate model nanoparticle configurations using reverse Monte Carlo simulations, which illustrate sequence-dependence in both surface structure and surface composition. Replica exchange solute tempering molecular dynamic simulations were also used to predict the modes of peptide binding on monometallic surfaces, indicating that different sequences bind to the metal interfaces via different mechanisms. As a testbed reaction, electrocatalytic methanol oxidation experiments were performed, wherein differences in catalytic activity are clearly observed in materials with identical bimetallic composition. Finally, taken together, this study indicates that peptides could be used to arrive at bimetallic surfaces with enhanced catalytic properties, which could be leveraged for rational bimetallic nanoparticle design using peptide-enabled approaches.« less

Peptide-Directed PdAu Nanoscale Surface Segregation: Toward Controlled Bimetallic Architecture for Catalytic Materials

DOE PAGES

Bedford, Nicholas M.; Showalter, Allison R.; Woehl, Taylor J.; ...

2016-09-01

Bimetallic nanoparticles are of immense scientific and technological interest given the synergistic properties observed when mixing two different metallic species at the nanoscale. This is particularly prevalent in catalysis, where bimetallic nanoparticles often exhibit improved catalytic activity and durability over their monometallic counterparts. Yet despite intense research efforts, little is understood regarding how to optimize bimetallic surface composition and structure synthetically using rational design principles. Recently, it has been demonstrated that peptide-enabled routes for nanoparticle synthesis result in materials with sequence-dependent catalytic properties, providing an opportunity for rational design through sequence manipulation. In this study, bimetallic PdAu nanoparticles are synthesizedmore » with a small set of peptides containing known Pd and Au binding motifs. The resulting nanoparticles were extensively characterized using high-resolution scanning transmission electron microscopy, X-ray absorption spectroscopy and high-energy X-ray diffraction coupled to atomic pair distribution function analysis. Structural information obtained from synchrotron radiation methods were then used to generate model nanoparticle configurations using reverse Monte Carlo simulations, which illustrate sequence-dependence in both surface structure and surface composition. Replica exchange solute tempering molecular dynamic simulations were also used to predict the modes of peptide binding on monometallic surfaces, indicating that different sequences bind to the metal interfaces via different mechanisms. As a testbed reaction, electrocatalytic methanol oxidation experiments were performed, wherein differences in catalytic activity are clearly observed in materials with identical bimetallic composition. Finally, taken together, this study indicates that peptides could be used to arrive at bimetallic surfaces with enhanced catalytic properties, which could be leveraged for rational bimetallic nanoparticle design using peptide-enabled approaches.« less

MoRFPred-plus: Computational Identification of MoRFs in Protein Sequences using Physicochemical Properties and HMM profiles.

PubMed

Sharma, Ronesh; Bayarjargal, Maitsetseg; Tsunoda, Tatsuhiko; Patil, Ashwini; Sharma, Alok

2018-01-21

Intrinsically Disordered Proteins (IDPs) lack stable tertiary structure and they actively participate in performing various biological functions. These IDPs expose short binding regions called Molecular Recognition Features (MoRFs) that permit interaction with structured protein regions. Upon interaction they undergo a disorder-to-order transition as a result of which their functionality arises. Predicting these MoRFs in disordered protein sequences is a challenging task. In this study, we present MoRFpred-plus, an improved predictor over our previous proposed predictor to identify MoRFs in disordered protein sequences. Two separate independent propensity scores are computed via incorporating physicochemical properties and HMM profiles, these scores are combined to predict final MoRF propensity score for a given residue. The first score reflects the characteristics of a query residue to be part of MoRF region based on the composition and similarity of assumed MoRF and flank regions. The second score reflects the characteristics of a query residue to be part of MoRF region based on the properties of flanks associated around the given residue in the query protein sequence. The propensity scores are processed and common averaging is applied to generate the final prediction score of MoRFpred-plus. Performance of the proposed predictor is compared with available MoRF predictors, MoRFchibi, MoRFpred, and ANCHOR. Using previously collected training and test sets used to evaluate the mentioned predictors, the proposed predictor outperforms these predictors and generates lower false positive rate. In addition, MoRFpred-plus is a downloadable predictor, which makes it useful as it can be used as input to other computational tools. https://github.com/roneshsharma/MoRFpred-plus/wiki/MoRFpred-plus:-Download. Copyright © 2017 Elsevier Ltd. All rights reserved.

THE YOUNG OPEN CLUSTERS KING 12, NGC 7788, AND NGC 7790: PRE-MAIN-SEQUENCE STARS AND EXTENDED STELLAR HALOS

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

Davidge, T. J.

2012-12-20

The stellar contents of the open clusters King 12, NGC 7788, and NGC 7790 are investigated using MegaCam images. Comparisons with isochrones yield an age <20 Myr for King 12, 20-40 Myr for NGC 7788, and 60-80 Myr for NGC 7790 based on the properties of stars near the main-sequence turnoff (MSTO) in each cluster. The reddening of NGC 7788 is much larger than previously estimated. The luminosity functions (LFs) of King 12 and NGC 7788 show breaks that are attributed to the onset of pre-main-sequence (PMS) objects, and comparisons with models of PMS evolution yield ages that are consistentmore » with those measured from stars near the MSTO. In contrast, the r' LF of main-sequence stars in NGC 7790 is matched to r' = 20 by a model that is based on the solar neighborhood mass function. The structural properties of all three clusters are investigated by examining the two-point angular correlation function of blue main-sequence stars. King 12 and NGC 7788 are each surrounded by a stellar halo that extends out to a radius of 5 arcmin ({approx}3.4 pc). It is suggested that these halos form in response to large-scale mass ejection early in the evolution of the clusters, as predicted by models. In contrast, blue main-sequence stars in NGC 7790 are traced out to a radius of {approx}7.5 arcmin ({approx}5.5 pc), with no evidence of a halo. It is suggested that all three clusters may have originated in the same star-forming complex, but not in the same giant molecular cloud.« less

ZifBASE: a database of zinc finger proteins and associated resources.

PubMed

Jayakanthan, Mannu; Muthukumaran, Jayaraman; Chandrasekar, Sanniyasi; Chawla, Konika; Punetha, Ankita; Sundar, Durai

2009-09-09

Information on the occurrence of zinc finger protein motifs in genomes is crucial to the developing field of molecular genome engineering. The knowledge of their target DNA-binding sequences is vital to develop chimeric proteins for targeted genome engineering and site-specific gene correction. There is a need to develop a computational resource of zinc finger proteins (ZFP) to identify the potential binding sites and its location, which reduce the time of in vivo task, and overcome the difficulties in selecting the specific type of zinc finger protein and the target site in the DNA sequence. ZifBASE provides an extensive collection of various natural and engineered ZFP. It uses standard names and a genetic and structural classification scheme to present data retrieved from UniProtKB, GenBank, Protein Data Bank, ModBase, Protein Model Portal and the literature. It also incorporates specialized features of ZFP including finger sequences and positions, number of fingers, physiochemical properties, classes, framework, PubMed citations with links to experimental structures (PDB, if available) and modeled structures of natural zinc finger proteins. ZifBASE provides information on zinc finger proteins (both natural and engineered ones), the number of finger units in each of the zinc finger proteins (with multiple fingers), the synergy between the adjacent fingers and their positions. Additionally, it gives the individual finger sequence and their target DNA site to which it binds for better and clear understanding on the interactions of adjacent fingers. The current version of ZifBASE contains 139 entries of which 89 are engineered ZFPs, containing 3-7F totaling to 296 fingers. There are 50 natural zinc finger protein entries ranging from 2-13F, totaling to 307 fingers. It has sequences and structures from literature, Protein Data Bank, ModBase and Protein Model Portal. The interface is cross linked to other public databases like UniprotKB, PDB, ModBase and Protein Model Portal and PubMed for making it more informative. A database is established to maintain the information of the sequence features, including the class, framework, number of fingers, residues, position, recognition site and physio-chemical properties (molecular weight, isoelectric point) of both natural and engineered zinc finger proteins and dissociation constant of few. ZifBASE can provide more effective and efficient way of accessing the zinc finger protein sequences and their target binding sites with the links to their three-dimensional structures. All the data and functions are available at the advanced web-based search interface http://web.iitd.ac.in/~sundar/zifbase.

A configuration space of homologous proteins conserving mutual information and allowing a phylogeny inference based on pair-wise Z-score probabilities.

PubMed

Bastien, Olivier; Ortet, Philippe; Roy, Sylvaine; Maréchal, Eric

2005-03-10

Popular methods to reconstruct molecular phylogenies are based on multiple sequence alignments, in which addition or removal of data may change the resulting tree topology. We have sought a representation of homologous proteins that would conserve the information of pair-wise sequence alignments, respect probabilistic properties of Z-scores (Monte Carlo methods applied to pair-wise comparisons) and be the basis for a novel method of consistent and stable phylogenetic reconstruction. We have built up a spatial representation of protein sequences using concepts from particle physics (configuration space) and respecting a frame of constraints deduced from pair-wise alignment score properties in information theory. The obtained configuration space of homologous proteins (CSHP) allows the representation of real and shuffled sequences, and thereupon an expression of the TULIP theorem for Z-score probabilities. Based on the CSHP, we propose a phylogeny reconstruction using Z-scores. Deduced trees, called TULIP trees, are consistent with multiple-alignment based trees. Furthermore, the TULIP tree reconstruction method provides a solution for some previously reported incongruent results, such as the apicomplexan enolase phylogeny. The CSHP is a unified model that conserves mutual information between proteins in the way physical models conserve energy. Applications include the reconstruction of evolutionary consistent and robust trees, the topology of which is based on a spatial representation that is not reordered after addition or removal of sequences. The CSHP and its assigned phylogenetic topology, provide a powerful and easily updated representation for massive pair-wise genome comparisons based on Z-score computations.

Probing the electrostatics and pharmacologic modulation of sequence-specific binding by the DNA-binding domain of the ETS-family transcription factor PU.1: a binding affinity and kinetics investigation

PubMed Central

Munde, Manoj; Poon, Gregory M. K.; Wilson, W. David

2013-01-01

Members of the ETS family of transcription factors regulate a functionally diverse array of genes. All ETS proteins share a structurally-conserved but sequence-divergent DNA-binding domain, known as the ETS domain. Although the structure and thermodynamics of the ETS-DNA complexes are well known, little is known about the kinetics of sequence recognition, a facet that offers potential insight into its molecular mechanism. We have characterized DNA binding by the ETS domain of PU.1 by biosensor-surface plasmon resonance (SPR). SPR analysis revealed a striking kinetic profile for DNA binding by the PU.1 ETS domain. At low salt concentrations, it binds high-affinity cognate DNA with a very slow association rate constant (≤105 M−1 s−1), compensated by a correspondingly small dissociation rate constant. The kinetics are strongly salt-dependent but mutually balance to produce a relatively weak dependence in the equilibrium constant. This profile contrasts sharply with reported data for other ETS domains (e.g., Ets-1, TEL) for which high-affinity binding is driven by rapid association (>107 M−1 s−1). We interpret this difference in terms of the hydration properties of ETS-DNA binding and propose that at least two mechanisms of sequence recognition are employed by this family of DNA-binding domain. Additionally, we use SPR to demonstrate the potential for pharmacological inhibition of sequence-specific ETS-DNA binding, using the minor groove-binding distamycin as a model compound. Our work establishes SPR as a valuable technique for extending our understanding of the molecular mechanisms of ETS-DNA interactions as well as developing potential small-molecule agents for biotechnological and therapeutic purposes. PMID:23416556

Purification of an alpha amylase from Aspergillus flavus NSH9 and molecular characterization of its nucleotide gene sequence.

PubMed

Karim, Kazi Muhammad Rezaul; Husaini, Ahmad; Sing, Ngieng Ngui; Sinang, Fazia Mohd; Roslan, Hairul Azman; Hussain, Hasnain

2018-04-01

In this study, an alpha-amylase enzyme from a locally isolated Aspergillus flavus NSH9 was purified and characterized. The extracellular α-amylase was purified by ammonium sulfate precipitation and anion-exchange chromatography at a final yield of 2.55-fold and recovery of 11.73%. The molecular mass of the purified α-amylase was estimated to be 54 kDa using SDS-PAGE and the enzyme exhibited optimal catalytic activity at pH 5.0 and temperature of 50 °C. The enzyme was also thermally stable at 50 °C, with 87% residual activity after 60 min. As a metalloenzymes containing calcium, the purified α-amylase showed significantly increased enzyme activity in the presence of Ca 2+ ions. Further gene isolation and characterization shows that the α-amylase gene of A. flavus NSH9 contained eight introns and an open reading frame that encodes for 499 amino acids with the first 21 amino acids presumed to be a signal peptide. Analysis of the deduced peptide sequence showed the presence of three conserved catalytic residues of α-amylase, two Ca 2+ -binding sites, seven conserved peptide sequences, and several other properties that indicates the protein belongs to glycosyl hydrolase family 13 capable of acting on α-1,4-bonds only. Based on sequence similarity, the deduced peptide sequence of A. flavus NSH9 α-amylase was also found to carry two potential surface/secondary-binding site (SBS) residues (Trp 237 and Tyr 409) that might be playing crucial roles in both the enzyme activity and also the binding of starch granules.

Sequence- and Temperature-Dependent Properties of Unfolded and Disordered Proteins from Atomistic Simulations.

PubMed

Zerze, Gül H; Best, Robert B; Mittal, Jeetain

2015-11-19

We use all-atom molecular simulation with explicit solvent to study the properties of selected intrinsically disordered proteins and unfolded states of foldable proteins, which include chain dimensions and shape, secondary structure propensity, solvent accessible surface area, and contact formation. We find that the qualitative scaling behavior of the chains matches expectations from theory under ambient conditions. In particular, unfolded globular proteins tend to be more collapsed under the same conditions than charged disordered sequences of the same length. However, inclusion of explicit solvent in addition naturally captures temperature-dependent solvation effects, which results in an initial collapse of the chains as temperature is increased, in qualitative agreement with experiment. There is a universal origin to the collapse, revealed in the change of hydration of individual residues as a function of temperature: namely, that the initial collapse is driven by unfavorable solvation free energy of individual residues, which in turn has a strong temperature dependence. We also observe that in unfolded globular proteins, increased temperature also initially favors formation of native-like (rather than non-native-like) structure. Our results help to establish how sequence encodes the degree of intrinsic disorder or order as well as its response to changes in environmental conditions.

Comparative modeling without implicit sequence alignments.

PubMed

Kolinski, Andrzej; Gront, Dominik

2007-10-01

The number of known protein sequences is about thousand times larger than the number of experimentally solved 3D structures. For more than half of the protein sequences a close or distant structural analog could be identified. The key starting point in a classical comparative modeling is to generate the best possible sequence alignment with a template or templates. With decreasing sequence similarity, the number of errors in the alignments increases and these errors are the main causes of the decreasing accuracy of the molecular models generated. Here we propose a new approach to comparative modeling, which does not require the implicit alignment - the model building phase explores geometric, evolutionary and physical properties of a template (or templates). The proposed method requires prior identification of a template, although the initial sequence alignment is ignored. The model is built using a very efficient reduced representation search engine CABS to find the best possible superposition of the query protein onto the template represented as a 3D multi-featured scaffold. The criteria used include: sequence similarity, predicted secondary structure consistency, local geometric features and hydrophobicity profile. For more difficult cases, the new method qualitatively outperforms existing schemes of comparative modeling. The algorithm unifies de novo modeling, 3D threading and sequence-based methods. The main idea is general and could be easily combined with other efficient modeling tools as Rosetta, UNRES and others.

Solid phase sequencing of double-stranded nucleic acids

DOEpatents

Fu, Dong-Jing; Cantor, Charles R.; Koster, Hubert; Smith, Cassandra L.

2002-01-01

This invention relates to methods for detecting and sequencing of target double-stranded nucleic acid sequences, to nucleic acid probes and arrays of probes useful in these methods, and to kits and systems which contain these probes. Useful methods involve hybridizing the nucleic acids or nucleic acids which represent complementary or homologous sequences of the target to an array of nucleic acid probes. These probe comprise a single-stranded portion, an optional double-stranded portion and a variable sequence within the single-stranded portion. The molecular weights of the hybridized nucleic acids of the set can be determined by mass spectroscopy, and the sequence of the target determined from the molecular weights of the fragments. Nucleic acids whose sequences can be determined include nucleic acids in biological samples such as patient biopsies and environmental samples. Probes may be fixed to a solid support such as a hybridization chip to facilitate automated determination of molecular weights and identification of the target sequence.

Information theory applications for biological sequence analysis.

PubMed

Vinga, Susana

2014-05-01

Information theory (IT) addresses the analysis of communication systems and has been widely applied in molecular biology. In particular, alignment-free sequence analysis and comparison greatly benefited from concepts derived from IT, such as entropy and mutual information. This review covers several aspects of IT applications, ranging from genome global analysis and comparison, including block-entropy estimation and resolution-free metrics based on iterative maps, to local analysis, comprising the classification of motifs, prediction of transcription factor binding sites and sequence characterization based on linguistic complexity and entropic profiles. IT has also been applied to high-level correlations that combine DNA, RNA or protein features with sequence-independent properties, such as gene mapping and phenotype analysis, and has also provided models based on communication systems theory to describe information transmission channels at the cell level and also during evolutionary processes. While not exhaustive, this review attempts to categorize existing methods and to indicate their relation with broader transversal topics such as genomic signatures, data compression and complexity, time series analysis and phylogenetic classification, providing a resource for future developments in this promising area.

A novel, extremely alkaliphilic and cold-active esterase from Antarctic desert soil.

PubMed

Hu, Xiao Ping; Heath, Caroline; Taylor, Mark Paul; Tuffin, Marla; Cowan, Don

2012-01-01

A novel, cold-active and highly alkaliphilic esterase was isolated from an Antarctic desert soil metagenomic library by functional screening. The 1,044 bp gene sequence contained several conserved regions common to lipases/esterases, but lacked clear classification based on sequence analysis alone. Moderate (<40%) amino acid sequence similarity to known esterases was apparent (the closest neighbour being a hypothetical protein from Chitinophaga pinensis), despite phylogenetic distance to many of the lipolytic "families". The enzyme functionally demonstrated activity towards shorter chain p-nitrophenyl esters with the optimal activity recorded towards p-nitrophenyl propionate (C3). The enzyme possessed an apparent T(opt) at 20°C and a pH optimum at pH 11. Esterases possessing such extreme alkaliphily are rare and so this enzyme represents an intriguing novel locus in protein sequence space. A metagenomic approach has been shown, in this case, to yield an enzyme with quite different sequential/structural properties to known lipases. It serves as an excellent candidate for analysis of the molecular mechanisms responsible for both cold and alkaline activity and novel structure-function relationships of esterase activity.

Characterization of Histone H2A Derived Antimicrobial Peptides, Harriottins, from Sicklefin Chimaera Neoharriotta pinnata (Schnakenbeck, 1931) and Its Evolutionary Divergence with respect to CO1 and Histone H2A.

PubMed

Sathyan, Naveen; Philip, Rosamma; Chaithanya, E R; Anil Kumar, P R; Sanjeevan, V N; Singh, I S Bright

2013-01-01

Antimicrobial peptides (AMPs) are humoral innate immune components of fishes that provide protection against pathogenic infections. Histone derived antimicrobial peptides are reported to actively participate in the immune defenses of fishes. Present study deals with identification of putative antimicrobial sequences from the histone H2A of sicklefin chimaera, Neoharriotta pinnata. A 52 amino acid residue termed Harriottin-1, a 40 amino acid Harriottin-2, and a 21 mer Harriottin-3 were identified to possess antimicrobial sequence motif. Physicochemical properties and molecular structure of Harriottins are in agreement with the characteristic features of antimicrobial peptides, indicating its potential role in innate immunity of sicklefin chimaera. The histone H2A sequence of sicklefin chimera was found to differ from previously reported histone H2A sequences. Phylogenetic analysis based on histone H2A and cytochrome oxidase subunit-1 (CO1) gene revealed N. pinnata to occupy an intermediate position with respect to invertebrates and vertebrates.

Characterization of Histone H2A Derived Antimicrobial Peptides, Harriottins, from Sicklefin Chimaera Neoharriotta pinnata (Schnakenbeck, 1931) and Its Evolutionary Divergence with respect to CO1 and Histone H2A

PubMed Central

Sathyan, Naveen; Philip, Rosamma; Chaithanya, E. R.; Anil Kumar, P. R.; Sanjeevan, V. N.; Singh, I. S. Bright

2013-01-01

Antimicrobial peptides (AMPs) are humoral innate immune components of fishes that provide protection against pathogenic infections. Histone derived antimicrobial peptides are reported to actively participate in the immune defenses of fishes. Present study deals with identification of putative antimicrobial sequences from the histone H2A of sicklefin chimaera, Neoharriotta pinnata. A 52 amino acid residue termed Harriottin-1, a 40 amino acid Harriottin-2, and a 21 mer Harriottin-3 were identified to possess antimicrobial sequence motif. Physicochemical properties and molecular structure of Harriottins are in agreement with the characteristic features of antimicrobial peptides, indicating its potential role in innate immunity of sicklefin chimaera. The histone H2A sequence of sicklefin chimera was found to differ from previously reported histone H2A sequences. Phylogenetic analysis based on histone H2A and cytochrome oxidase subunit-1 (CO1) gene revealed N. pinnata to occupy an intermediate position with respect to invertebrates and vertebrates. PMID:27398241

PCV: An Alignment Free Method for Finding Homologous Nucleotide Sequences and its Application in Phylogenetic Study.

PubMed

Kumar, Rajnish; Mishra, Bharat Kumar; Lahiri, Tapobrata; Kumar, Gautam; Kumar, Nilesh; Gupta, Rahul; Pal, Manoj Kumar

2017-06-01

Online retrieval of the homologous nucleotide sequences through existing alignment techniques is a common practice against the given database of sequences. The salient point of these techniques is their dependence on local alignment techniques and scoring matrices the reliability of which is limited by computational complexity and accuracy. Toward this direction, this work offers a novel way for numerical representation of genes which can further help in dividing the data space into smaller partitions helping formation of a search tree. In this context, this paper introduces a 36-dimensional Periodicity Count Value (PCV) which is representative of a particular nucleotide sequence and created through adaptation from the concept of stochastic model of Kolekar et al. (American Institute of Physics 1298:307-312, 2010. doi: 10.1063/1.3516320 ). The PCV construct uses information on physicochemical properties of nucleotides and their positional distribution pattern within a gene. It is observed that PCV representation of gene reduces computational cost in the calculation of distances between a pair of genes while being consistent with the existing methods. The validity of PCV-based method was further tested through their use in molecular phylogeny constructs in comparison with that using existing sequence alignment methods.

Genome and transcriptome of the regeneration-competent flatworm, Macrostomum lignano.

PubMed

Wasik, Kaja; Gurtowski, James; Zhou, Xin; Ramos, Olivia Mendivil; Delás, M Joaquina; Battistoni, Giorgia; El Demerdash, Osama; Falciatori, Ilaria; Vizoso, Dita B; Smith, Andrew D; Ladurner, Peter; Schärer, Lukas; McCombie, W Richard; Hannon, Gregory J; Schatz, Michael

2015-10-06

The free-living flatworm, Macrostomum lignano has an impressive regenerative capacity. Following injury, it can regenerate almost an entirely new organism because of the presence of an abundant somatic stem cell population, the neoblasts. This set of unique properties makes many flatworms attractive organisms for studying the evolution of pathways involved in tissue self-renewal, cell-fate specification, and regeneration. The use of these organisms as models, however, is hampered by the lack of a well-assembled and annotated genome sequences, fundamental to modern genetic and molecular studies. Here we report the genomic sequence of M. lignano and an accompanying characterization of its transcriptome. The genome structure of M. lignano is remarkably complex, with ∼75% of its sequence being comprised of simple repeats and transposon sequences. This has made high-quality assembly from Illumina reads alone impossible (N50=222 bp). We therefore generated 130× coverage by long sequencing reads from the Pacific Biosciences platform to create a substantially improved assembly with an N50 of 64 Kbp. We complemented the reference genome with an assembled and annotated transcriptome, and used both of these datasets in combination to probe gene-expression patterns during regeneration, examining pathways important to stem cell function.

De Novo Assembly of Auricularia polytricha Transcriptome Using Illumina Sequencing for Gene Discovery and SSR Marker Identification

PubMed Central

Zhou, Yan; Chen, Lianfu; Fan, Xiuzhi; Bian, Yinbing

2014-01-01

Auricularia polytricha (Mont.) Sacc., a type of edible black-brown mushroom with a gelatinous and modality-specific fruiting body, is in high demand in Asia due to its nutritional and medicinal properties. Illumina Solexa sequenceing technology was used to generate very large transcript sequences from the mycelium and the mature fruiting body of A. polytricha for gene discovery and molecular marker development. De novo assembly generated 36,483 ESTs with an N50 length of 636 bp. A total of 28,108 ESTs demonstrated significant hits with known proteins in the nr database, and 94.03% of the annotated ESTs showed the greatest similarity to A. delicata, a related species of A. polytricha. Functional categorization of the Gene Ontology (GO), Clusters of Orthologous Groups (COG) and Kyoto Encyclopedia of Genes and Genomes (KEGG) metabolic pathways revealed the conservation of genes involved in various biological processes in A. polytricha. Gene expression profile analysis indicated that a total of 2,057 ESTs were differentially expressed, including 1,020 ESTs that were up-regulated in the mycelium and 1,037 up-regulated in the fruiting body. Functional enrichment showed that the ESTs associated with biosynthesis, metabolism and assembly of proteins were more active in fruiting body development. The expression patterns of homologous transcription factors indicated that the molecular mechanisms of fruiting body formation and development were not exactly the same as for other agarics. Interestingly, an EST encoding tyrosinase was significantly up-regulated in the fruiting body, indicating that melanins accumulated during the processes of the formation of the black-brown color of the fruiting body in A. polytricha development. In addition, a total of 1,715 potential SSRs were detected in this transcriptome. The transcriptome analysis of A. polytricha provides valuable sequence resources and numerous molecular markers to facilitate further functional genomics studies and genetic researches on this fungus. PMID:24626227

Molecular cloning, expression and characterization of Pru a 1, the major cherry allergen.

PubMed

Scheurer, S; Metzner, K; Haustein, D; Vieths, S

1997-06-01

A high percentage of birch pollen allergic patients experiences food hypersensitivity reactions after ingestion of several fruits and vegetables. Previous work demonstrated common epitopes on an allergen of Mr 18,000 from sweet cherry (Prunus avium) and Bet v 1, the major allergen from birch pollen. N-terminal amino acid sequencing showed a sequence identity of 67% with Bet v 1. Here we report the cloning and cDNA sequencing of this cherry allergen. The entire deduced amino acid sequence described a protein of Mr 17,700 with 59.1% identity to Bet v 1. High degrees of identity in the range of 40 to 60% were also found with related allergens from other kinds of tree pollen and plant foods as well as with stress-induced proteins from food plants such as parsley, potato and soya. The coding DNA of the cherry protein was cloned into vector pET-16b and expressed in E. coli strain BL21(DE3) as a His-tag fusion protein. As shown by SDS-PAGE, the apparent molecular masses of the nonfusion protein and the natural allergen were identical. The fusion protein showed high IgE binding potency when sera from patients allergic to cherry were tested by immunoblotting and enzyme allergosorbent tests. Moreover, it cross-reacted strongly with IgE specific for the natural counterpart and for Bet v 1. The high biological activity of the recombinant fusion protein was further confirmed by the induction of a strong histamine release in basophils from cherry-allergic patients. Since sera from 17/19 of such patients contained IgE against this allergen it was classified as a major allergen and named Pru a 1. Recombinant Pru a 1 mimics most of the allergenic potency of cherry extract and hence could be a useful tool for studying the molecular and immunological properties of pollen related food allergens.

Trans-Homolog Interactions Facilitating Paramutation in Maize

PubMed Central

2015-01-01

Paramutations represent locus-specific trans-homolog interactions affecting the heritable silencing properties of endogenous alleles. Although examples of paramutation are well studied in maize (Zea mays), the responsible mechanisms remain unclear. Genetic analyses indicate roles for plant-specific DNA-dependent RNA polymerases that generate small RNAs, and current working models hypothesize that these small RNAs direct heritable changes at sequences often acting as transcriptional enhancers. Several studies have defined specific sequences that mediate paramutation behaviors, and recent results identify a diversity of DNA-dependent RNA polymerase complexes operating in maize. Other reports ascribe broader roles for some of these complexes in normal genome function. This review highlights recent research to understand the molecular mechanisms of paramutation and examines evidence relevant to small RNA-based modes of transgenerational epigenetic inheritance. PMID:26149572

Advances in Omics and Bioinformatics Tools for Systems Analyses of Plant Functions

PubMed Central

Mochida, Keiichi; Shinozaki, Kazuo

2011-01-01

Omics and bioinformatics are essential to understanding the molecular systems that underlie various plant functions. Recent game-changing sequencing technologies have revitalized sequencing approaches in genomics and have produced opportunities for various emerging analytical applications. Driven by technological advances, several new omics layers such as the interactome, epigenome and hormonome have emerged. Furthermore, in several plant species, the development of omics resources has progressed to address particular biological properties of individual species. Integration of knowledge from omics-based research is an emerging issue as researchers seek to identify significance, gain biological insights and promote translational research. From these perspectives, we provide this review of the emerging aspects of plant systems research based on omics and bioinformatics analyses together with their associated resources and technological advances. PMID:22156726

[Progress on molecular biology of Isaria farinosa, pathogen of host of Ophiocordyceps sinensis during the artificial culture].

PubMed

Liu, Fei; Wu, Xiao-Li; Liu, Ying; Chen, Da-Xia; Zhang, De-Li; Yang, Da-Jian

2016-02-01

Isaria farinosa is the pathogen of the host of Ophiocordyceps sinensis. The present research has analyzed the progress on the molecular biology according to the bibliometrics, the sequences (including the gene sequences) of I. farinosa in the NCBI. The results indicated that different country had published different number of the papers, and had landed different kinds and different number of the sequences (including the gene sequences). China had published the most number of the papers, and had landed the most number of the sequences (including the gene sequences). America had landed the most numbers of the function genes. The main content about the pathogen study was focus on the biological controlling. The main content about the molecular study concentrated on the phylogenies classification. In recent years some protease genes and chitinase genes had been researched. With the increase of the effect on the healthy of O. sinensis, and the whole sequence and more and more pharmacological activities of I. farinosa being made known to the public, the study on the molecular biology of the I. farinosa would be deeper and wider. Copyright© by the Chinese Pharmaceutical Association.

Molecular modeling of the elastomeric properties of repeating units and building blocks of resilin, a disordered elastic protein.

PubMed

Khandaker, Md Shahriar K; Dudek, Daniel M; Beers, Eric P; Dillard, David A; Bevan, David R

2016-08-01

The mechanisms responsible for the properties of disordered elastomeric proteins are not well known. To better understand the relationship between elastomeric behavior and amino acid sequence, we investigated resilin, a disordered rubber-like protein, found in specialized regions of the cuticle of insects. Resilin of Drosophila melanogaster contains Gly-rich repetitive motifs comprised of the amino acids, PSSSYGAPGGGNGGR, which confer elastic properties to resilin. The repetitive motifs of insect resilin can be divided into smaller partially conserved building blocks: PSS, SYGAP, GGGN and GGR. Using molecular dynamics (MD) simulations, we studied the relative roles of SYGAP, and its less common variants SYSAP and TYGAP, on the elastomeric properties of resilin. Results showed that SYGAP adopts a bent structure that is one-half to one-third the end-to-end length of the other motifs having an equal number of amino acids but containing SYSAP or TYGAP substituted for SYGAP. The bent structure of SYGAP forms due to conformational freedom of glycine, and hydrogen bonding within the motif apparently plays a role in maintaining this conformation. These structural features of SYGAP result in higher extensibility compared to other motifs, which may contribute to elastic properties at the macroscopic level. Overall, the results are consistent with a role for the SYGAP building block in the elastomeric properties of these disordered proteins. What we learned from simulating the repetitive motifs of resilin may be applicable to the biology and mechanics of other elastomeric biomaterials, and may provide us the deeper understanding of their unique properties. Copyright © 2016 Elsevier Ltd. All rights reserved.

High-throughput sequencing of black pepper root transcriptome.

PubMed

Gordo, Sheila M C; Pinheiro, Daniel G; Moreira, Edith C O; Rodrigues, Simone M; Poltronieri, Marli C; de Lemos, Oriel F; da Silva, Israel Tojal; Ramos, Rommel T J; Silva, Artur; Schneider, Horacio; Silva, Wilson A; Sampaio, Iracilda; Darnet, Sylvain

2012-09-17

Black pepper (Piper nigrum L.) is one of the most popular spices in the world. It is used in cooking and the preservation of food and even has medicinal properties. Losses in production from disease are a major limitation in the culture of this crop. The major diseases are root rot and foot rot, which are results of root infection by Fusarium solani and Phytophtora capsici, respectively. Understanding the molecular interaction between the pathogens and the host's root region is important for obtaining resistant cultivars by biotechnological breeding. Genetic and molecular data for this species, though, are limited. In this paper, RNA-Seq technology has been employed, for the first time, to describe the root transcriptome of black pepper. The root transcriptome of black pepper was sequenced by the NGS SOLiD platform and assembled using the multiple-k method. Blast2Go and orthoMCL methods were used to annotate 10338 unigenes. The 4472 predicted proteins showed about 52% homology with the Arabidopsis proteome. Two root proteomes identified 615 proteins, which seem to define the plant's root pattern. Simple-sequence repeats were identified that may be useful in studies of genetic diversity and may have applications in biotechnology and ecology. This dataset of 10338 unigenes is crucially important for the biotechnological breeding of black pepper and the ecogenomics of the Magnoliids, a major group of basal angiosperms.

High-throughput sequencing of black pepper root transcriptome

PubMed Central

2012-01-01

Background Black pepper (Piper nigrum L.) is one of the most popular spices in the world. It is used in cooking and the preservation of food and even has medicinal properties. Losses in production from disease are a major limitation in the culture of this crop. The major diseases are root rot and foot rot, which are results of root infection by Fusarium solani and Phytophtora capsici, respectively. Understanding the molecular interaction between the pathogens and the host’s root region is important for obtaining resistant cultivars by biotechnological breeding. Genetic and molecular data for this species, though, are limited. In this paper, RNA-Seq technology has been employed, for the first time, to describe the root transcriptome of black pepper. Results The root transcriptome of black pepper was sequenced by the NGS SOLiD platform and assembled using the multiple-k method. Blast2Go and orthoMCL methods were used to annotate 10338 unigenes. The 4472 predicted proteins showed about 52% homology with the Arabidopsis proteome. Two root proteomes identified 615 proteins, which seem to define the plant’s root pattern. Simple-sequence repeats were identified that may be useful in studies of genetic diversity and may have applications in biotechnology and ecology. Conclusions This dataset of 10338 unigenes is crucially important for the biotechnological breeding of black pepper and the ecogenomics of the Magnoliids, a major group of basal angiosperms. PMID:22984782

A safe an easy method for building consensus HIV sequences from 454 massively parallel sequencing data.

PubMed

Fernández-Caballero Rico, Jose Ángel; Chueca Porcuna, Natalia; Álvarez Estévez, Marta; Mosquera Gutiérrez, María Del Mar; Marcos Maeso, María Ángeles; García, Federico

2018-02-01

To show how to generate a consensus sequence from the information of massive parallel sequences data obtained from routine HIV anti-retroviral resistance studies, and that may be suitable for molecular epidemiology studies. Paired Sanger (Trugene-Siemens) and next-generation sequencing (NGS) (454 GSJunior-Roche) HIV RT and protease sequences from 62 patients were studied. NGS consensus sequences were generated using Mesquite, using 10%, 15%, and 20% thresholds. Molecular evolutionary genetics analysis (MEGA) was used for phylogenetic studies. At a 10% threshold, NGS-Sanger sequences from 17/62 patients were phylogenetically related, with a median bootstrap-value of 88% (IQR83.5-95.5). Association increased to 36/62 sequences, median bootstrap 94% (IQR85.5-98)], using a 15% threshold. Maximum association was at the 20% threshold, with 61/62 sequences associated, and a median bootstrap value of 99% (IQR98-100). A safe method is presented to generate consensus sequences from HIV-NGS data at 20% threshold, which will prove useful for molecular epidemiological studies. Copyright © 2016 Elsevier España, S.L.U. and Sociedad Española de Enfermedades Infecciosas y Microbiología Clínica. All rights reserved.

Teaching the Process of Molecular Phylogeny and Systematics: A Multi-Part Inquiry-Based Exercise

ERIC Educational Resources Information Center

Lents, Nathan H.; Cifuentes, Oscar E.; Carpi, Anthony

2010-01-01

Three approaches to molecular phylogenetics are demonstrated to biology students as they explore molecular data from "Homo sapiens" and four related primates. By analyzing DNA sequences, protein sequences, and chromosomal maps, students are repeatedly challenged to develop hypotheses regarding the ancestry of the five species. Although…

Towards Clinical Molecular Diagnosis of Inherited Cardiac Conditions: A Comparison of Bench-Top Genome DNA Sequencers

PubMed Central

Wilkinson, Samuel L.; John, Shibu; Walsh, Roddy; Novotny, Tomas; Valaskova, Iveta; Gupta, Manu; Game, Laurence; Barton, Paul J R.; Cook, Stuart A.; Ware, James S.

2013-01-01

Background Molecular genetic testing is recommended for diagnosis of inherited cardiac disease, to guide prognosis and treatment, but access is often limited by cost and availability. Recently introduced high-throughput bench-top DNA sequencing platforms have the potential to overcome these limitations. Methodology/Principal Findings We evaluated two next-generation sequencing (NGS) platforms for molecular diagnostics. The protein-coding regions of six genes associated with inherited arrhythmia syndromes were amplified from 15 human samples using parallelised multiplex PCR (Access Array, Fluidigm), and sequenced on the MiSeq (Illumina) and Ion Torrent PGM (Life Technologies). Overall, 97.9% of the target was sequenced adequately for variant calling on the MiSeq, and 96.8% on the Ion Torrent PGM. Regions missed tended to be of high GC-content, and most were problematic for both platforms. Variant calling was assessed using 107 variants detected using Sanger sequencing: within adequately sequenced regions, variant calling on both platforms was highly accurate (Sensitivity: MiSeq 100%, PGM 99.1%. Positive predictive value: MiSeq 95.9%, PGM 95.5%). At the time of the study the Ion Torrent PGM had a lower capital cost and individual runs were cheaper and faster. The MiSeq had a higher capacity (requiring fewer runs), with reduced hands-on time and simpler laboratory workflows. Both provide significant cost and time savings over conventional methods, even allowing for adjunct Sanger sequencing to validate findings and sequence exons missed by NGS. Conclusions/Significance MiSeq and Ion Torrent PGM both provide accurate variant detection as part of a PCR-based molecular diagnostic workflow, and provide alternative platforms for molecular diagnosis of inherited cardiac conditions. Though there were performance differences at this throughput, platforms differed primarily in terms of cost, scalability, protocol stability and ease of use. Compared with current molecular genetic diagnostic tests for inherited cardiac arrhythmias, these NGS approaches are faster, less expensive, and yet more comprehensive. PMID:23861798

Measuring the Electronic Properties of DNA-Specific Schottky Diodes Towards Detecting and Identifying Basidiomycetes DNA

PubMed Central

Periasamy, Vengadesh; Rizan, Nastaran; Al-Ta’ii, Hassan Maktuff Jaber; Tan, Yee Shin; Tajuddin, Hairul Annuar; Iwamoto, Mitsumasa

2016-01-01

The discovery of semiconducting behavior of deoxyribonucleic acid (DNA) has resulted in a large number of literatures in the study of DNA electronics. Sequence-specific electronic response provides a platform towards understanding charge transfer mechanism and therefore the electronic properties of DNA. It is possible to utilize these characteristic properties to identify/detect DNA. In this current work, we demonstrate a novel method of DNA-based identification of basidiomycetes using current-voltage (I-V) profiles obtained from DNA-specific Schottky barrier diodes. Electronic properties such as ideality factor, barrier height, shunt resistance, series resistance, turn-on voltage, knee-voltage, breakdown voltage and breakdown current were calculated and used to quantify the identification process as compared to morphological and molecular characterization techniques. The use of these techniques is necessary in order to study biodiversity, but sometimes it can be misleading and unreliable and is not sufficiently useful for the identification of fungi genera. Many of these methods have failed when it comes to identification of closely related species of certain genus like Pleurotus. Our electronics profiles, both in the negative and positive bias regions were however found to be highly characteristic according to the base-pair sequences. We believe that this simple, low-cost and practical method could be useful towards identifying and detecting DNA in biotechnology and pathology. PMID:27435636

Measuring the Electronic Properties of DNA-Specific Schottky Diodes Towards Detecting and Identifying Basidiomycetes DNA

NASA Astrophysics Data System (ADS)

Periasamy, Vengadesh; Rizan, Nastaran; Al-Ta'Ii, Hassan Maktuff Jaber; Tan, Yee Shin; Tajuddin, Hairul Annuar; Iwamoto, Mitsumasa

2016-07-01

The discovery of semiconducting behavior of deoxyribonucleic acid (DNA) has resulted in a large number of literatures in the study of DNA electronics. Sequence-specific electronic response provides a platform towards understanding charge transfer mechanism and therefore the electronic properties of DNA. It is possible to utilize these characteristic properties to identify/detect DNA. In this current work, we demonstrate a novel method of DNA-based identification of basidiomycetes using current-voltage (I-V) profiles obtained from DNA-specific Schottky barrier diodes. Electronic properties such as ideality factor, barrier height, shunt resistance, series resistance, turn-on voltage, knee-voltage, breakdown voltage and breakdown current were calculated and used to quantify the identification process as compared to morphological and molecular characterization techniques. The use of these techniques is necessary in order to study biodiversity, but sometimes it can be misleading and unreliable and is not sufficiently useful for the identification of fungi genera. Many of these methods have failed when it comes to identification of closely related species of certain genus like Pleurotus. Our electronics profiles, both in the negative and positive bias regions were however found to be highly characteristic according to the base-pair sequences. We believe that this simple, low-cost and practical method could be useful towards identifying and detecting DNA in biotechnology and pathology.

The Star-forming Main Sequence of Dwarf Low Surface Brightness Galaxies

NASA Astrophysics Data System (ADS)

McGaugh, Stacy S.; Schombert, James M.; Lelli, Federico

2017-12-01

We explore the star-forming properties of late-type, low surface brightness (LSB) galaxies. The star-forming main sequence ({SFR}-{M}* ) of LSB dwarfs has a steep slope, indistinguishable from unity (1.04 ± 0.06). They form a distinct sequence from more massive spirals, which exhibit a shallower slope. The break occurs around {M}* ≈ {10}10 {M}⊙ , and can also be seen in the gas mass—stellar mass plane. The global Kennicutt-Schmidt law ({SFR}-{M}g) has a slope of 1.47 ± 0.11 without the break seen in the main sequence. There is an ample supply of gas in LSB galaxies, which have gas depletion times well in excess of a Hubble time, and often tens of Hubble times. Only ˜ 3 % of this cold gas needs be in the form of molecular gas to sustain the observed star formation. In analogy with the faint, long-lived stars of the lower stellar main sequence, it may be appropriate to consider the main sequence of star-forming galaxies to be defined by thriving dwarfs (with {M}* < {10}10 {M}⊙ ), while massive spirals (with {M}* > {10}10 {M}⊙ ) are weary giants that constitute more of a turn-off population.

Easy design of colorimetric logic gates based on nonnatural base pairing and controlled assembly of gold nanoparticles.

PubMed

Zhang, Li; Wang, Zhong-Xia; Liang, Ru-Ping; Qiu, Jian-Ding

2013-07-16

Utilizing the principles of metal-ion-mediated base pairs (C-Ag-C and T-Hg-T), the pH-sensitive conformational transition of C-rich DNA strand, and the ligand-exchange process triggered by DL-dithiothreitol (DTT), a system of colorimetric logic gates (YES, AND, INHIBIT, and XOR) can be rationally constructed based on the aggregation of the DNA-modified Au NPs. The proposed logic operation system is simple, which consists of only T-/C-rich DNA-modified Au NPs, and it is unnecessary to exquisitely design and alter the DNA sequence for different multiple molecular logic operations. The nonnatural base pairing combined with unique optical properties of Au NPs promises great potential in multiplexed ion sensing, molecular-scale computers, and other computational logic devices.

INDUCTION OF RABBIT ANTIBODY WITH MOLECULAR UNIFORMITY AFTER IMMUNIZATION WITH GROUP C STREPTOCOCCI

PubMed Central

Eichmann, Klaus; Lackland, Henry; Hood, Leroy; Krause, Richard M.

1970-01-01

Antibodies with uniform properties may occur in rabbits after immunization with Group C streptococci. These precipitating antibodies possess specificity for the group-specific carbohydrate. Not uncommonly, their concentration is between 20 and 40 mg/ml of antiserum. Evidence for molecular uniformity in the case of one of these antibodies, described in detail here, includes: individual antigenic specificity; monodisperse distribution of the light chains by alkaline urea polyacrylamide disc electrophoresis; and a single amino acid in each of the first three N-terminal positions of the light chains. When the amino acid sequence of rabbit antibody b+ light chains (κ type) are aligned against their human κ counterparts, a definite homology is observed between the N-terminus of the human and the rabbit variable region. PMID:5409946

Molecular gas mass and star formation of 12 Virgo spiral galaxies along the ram pressure time sequence

NASA Astrophysics Data System (ADS)

Chung, Eun Jung; Kim, S.

2014-01-01

The ram pressure stripping is known as one of the most efficient mechanisms to deplete the ISM of a galaxy in the clusters of galaxies. As being affected continuously by ICM pressure, a galaxy may lose their gas that is the fuel of star formation, and consequently star formation rate would be changed. We select twelve Virgo spiral galaxies according to their stage of the ram pressure stripping event to probe possible consequences of star formation of spiral galaxies in the ram pressure and thus the evolution of galaxies in the Virgo cluster. We investigate the molecular gas properties, star formation activity, and gas depletion time along the time from the ram pressure peak. We also discussed the evolution of galaxies in the cluster.

Identification of Arcanobacterium pyogenes isolated by post mortem examinations of a bearded dragon and a gecko by phenotypic and genotypic properties.

PubMed

Ulbegi-Mohyla, H; Hijazin, M; Alber, J; Lämmler, C; Hassan, A A; Abdulmawjood, A; Prenger-Berninghoff, E; Weiss, R; Zschöck, M

2010-09-01

The present study was designed to identify phenotypically and genotypically two Arcanobacterium (A.) pyogenes strains isolated by post mortem examinations of a bearded dragon and a gecko. The A. pyogenes strains showed the typical biochemical properties and displayed CAMP-like synergistic hemolytic activities with various indicator strains. The species identity could be confirmed genotypically by amplification and sequencing of the 16S rDNA gene and, as novel target gene, by sequencing of the beta subunit of RNA polymerase encoding gene rpoB, of both strains and of reference strains representing nine species of the genus Arcanobacterium. The species identity of the two A. pyogenes strains could additionally be confirmed by PCR mediated amplification of species specific parts of the 16S-23S rDNA intergenic spacer region, the pyolysin encoding gene plo and by amplification of the collagen-binding protein encoding gene cbpA. All these molecular targets might help to improve the future identification and further characterization of A. pyogenes which, as demonstrated in the present study, could also be isolated from reptile specimens.

Using DNA-labelled nano- and microparticles to track particle transport in the environment

NASA Astrophysics Data System (ADS)

McNew, Coy; Wang, Chaozi; Dahlke, Helen; Lyon, Steve; Walter, Todd

2017-04-01

By utilizing bio-molecular nanotechnology developed for nano-medicines and drug delivery, we are able to produce DNA-labelled nano- and microparticle tracers for use in a myriad of environmental systems. The use of custom sequenced DNA allows for the fabrication of an enormous number of uniquely labelled tracers with identical transport properties (approximately 1.61 x 1060 unique sequences), each independently quantifiable, that can be applied simultaneously in any hydrologic system. By controlling the fabrication procedure to produce particles of custom size and charge, we are able to tag each size-charge combination uniquely in order to directly probe the effect of these variables on the transport properties of the particles. Here we present our methods for fabrication, extraction, and analysis of the DNA nano- and microparticle tracers, along with results from several successful applications of the tracers, including transport and retention analysis at the lab, continuum, and field scales. To date, our DNA-labelled nano- and microparticle tracers have proved useful in surface and subsurface water applications, soil retention, and even subglacial flow pathways. The range of potential applications continue to prove nearly limitless.

Role of mutation on fibril formation in small peptides by REMD

NASA Astrophysics Data System (ADS)

Mahmoudinobar, Farbod; Dias, Cristiano

Amyloid fibrils are now recognized as a common form of protein structure. They have wide implications for neurological diseases and entities involved in the survival of living organisms, e.g., silkmoth eggshells. Biological functions of these entities are often related to the superior mechanical strength of fibrils that persists over a broad range of chemical and thermal conditions desirable for various biotechnological applications, e.g., to encapsulate drugs. Mechanical properties of fibrils was shown to depend strongly on the amino acid sequence of its constituent peptides whereby bending rigidities can vary by two orders of magnitude. Therefore, the rational design of new fibril-prone peptides with tailored properties depends on our understanding of the relation between amino acid sequence and its propensity to fibrillize. In this presentation I will show results from extensive Replica Exchange Molecular Dynamics (REMD) simulations of a 12-residue peptide containing the fibril-prone motif KFFE and its mutants. Simulations are performed on monomers, dimers, and tetramers. I will discuss effects of side chain packing, hydrophobicity, charges and beta-sheet propensity on fibril formation. Physics Department, University Heights, Newark, New Jersey, 07102-1982, USA.

Links between plant and fungal communities across a deforestation chronosequence in the Amazon rainforest.

PubMed

Mueller, Rebecca C; Paula, Fabiana S; Mirza, Babur S; Rodrigues, Jorge L M; Nüsslein, Klaus; Bohannan, Brendan J M

2014-07-01

Understanding the interactions among microbial communities, plant communities and soil properties following deforestation could provide insights into the long-term effects of land-use change on ecosystem functions, and may help identify approaches that promote the recovery of degraded sites. We combined high-throughput sequencing of fungal rDNA and molecular barcoding of plant roots to estimate fungal and plant community composition in soil sampled across a chronosequence of deforestation. We found significant effects of land-use change on fungal community composition, which was more closely correlated to plant community composition than to changes in soil properties or geographic distance, providing evidence for strong links between above- and below-ground communities in tropical forests.

Cyclic mu-opioid receptor ligands containing multiple N-methylated amino acid residues.

PubMed

Adamska-Bartłomiejczyk, Anna; Janecka, Anna; Szabó, Márton Richárd; Cerlesi, Maria Camilla; Calo, Girolamo; Kluczyk, Alicja; Tömböly, Csaba; Borics, Attila

2017-04-15

In this study we report the in vitro activities of four cyclic opioid peptides with various sequence length/macrocycle size and N-methylamino acid residue content. N-Methylated amino acids were incorporated and cyclization was employed to enhance conformational rigidity to various extent. The effect of such modifications on ligand structure and binding properties were studied. The pentapeptide containing one endocyclic and one exocyclic N-methylated amino acid displayed the highest affinity to the mu-opioid receptor. This peptide was also shown to be a full agonist, while the other analogs failed to activate the mu opioid receptor. Results of molecular docking studies provided rationale for the explanation of binding properties on a structural basis. Copyright © 2017 Elsevier Ltd. All rights reserved.

Targeted Capture and High-Throughput Sequencing Using Molecular Inversion Probes (MIPs).

PubMed

Cantsilieris, Stuart; Stessman, Holly A; Shendure, Jay; Eichler, Evan E

2017-01-01

Molecular inversion probes (MIPs) in combination with massively parallel DNA sequencing represent a versatile, yet economical tool for targeted sequencing of genomic DNA. Several thousand genomic targets can be selectively captured using long oligonucleotides containing unique targeting arms and universal linkers. The ability to append sequencing adaptors and sample-specific barcodes allows large-scale pooling and subsequent high-throughput sequencing at relatively low cost per sample. Here, we describe a "wet bench" protocol detailing the capture and subsequent sequencing of >2000 genomic targets from 192 samples, representative of a single lane on the Illumina HiSeq 2000 platform.

Molecular diversity of arbuscular mycorrhizal fungi in relation to soil chemical properties and heavy metal contamination.

PubMed

Zarei, Mehdi; Hempel, Stefan; Wubet, Tesfaye; Schäfer, Tina; Savaghebi, Gholamreza; Jouzani, Gholamreza Salehi; Nekouei, Mojtaba Khayam; Buscot, François

2010-08-01

Abundance and diversity of arbuscular mycorrhizal fungi (AMF) associated with dominant plant species were studied along a transect from highly lead (Pb) and zinc (Zn) polluted to non-polluted soil at the Anguran open pit mine in Iran. Using an established primer set for AMF in the internal transcribed spacer (ITS) region of rDNA, nine different AMF sequence types were distinguished after phylogenetic analyses, showing remarkable differences in their distribution patterns along the transect. With decreasing Pb and Zn concentration, the number of AMF sequence types increased, however one sequence type was only found in the highly contaminated area. Multivariate statistical analysis revealed that further factors than HM soil concentration affect the AMF community at contaminated sites. Specifically, the soils' calcium carbonate equivalent and available P proved to be of importance, which illustrates that field studies on AMF distribution should also consider important environmental factors and their possible interactions. Copyright 2010 Elsevier Ltd. All rights reserved.

Landscape of Infiltrating T Cells in Liver Cancer Revealed by Single-Cell Sequencing.

PubMed

Zheng, Chunhong; Zheng, Liangtao; Yoo, Jae-Kwang; Guo, Huahu; Zhang, Yuanyuan; Guo, Xinyi; Kang, Boxi; Hu, Ruozhen; Huang, Julie Y; Zhang, Qiming; Liu, Zhouzerui; Dong, Minghui; Hu, Xueda; Ouyang, Wenjun; Peng, Jirun; Zhang, Zemin

2017-06-15

Systematic interrogation of tumor-infiltrating lymphocytes is key to the development of immunotherapies and the prediction of their clinical responses in cancers. Here, we perform deep single-cell RNA sequencing on 5,063 single T cells isolated from peripheral blood, tumor, and adjacent normal tissues from six hepatocellular carcinoma patients. The transcriptional profiles of these individual cells, coupled with assembled T cell receptor (TCR) sequences, enable us to identify 11 T cell subsets based on their molecular and functional properties and delineate their developmental trajectory. Specific subsets such as exhausted CD8 + T cells and Tregs are preferentially enriched and potentially clonally expanded in hepatocellular carcinoma (HCC), and we identified signature genes for each subset. One of the genes, layilin, is upregulated on activated CD8 + T cells and Tregs and represses the CD8 + T cell functions in vitro. This compendium of transcriptome data provides valuable insights and a rich resource for understanding the immune landscape in cancers. Copyright © 2017 Elsevier Inc. All rights reserved.

Controlling Self-Assembly of Engineered Peptides on Graphite by Rational Mutation

PubMed Central

So, Christopher R.; Hayamizu, Yuhei; Yazici, Hilal; Gresswell, Carolyn; Khatayevich, Dmitriy; Tamerler, Candan; Sarikaya, Mehmet

2012-01-01

Self-assembly of proteins on surfaces is utilized in many fields to integrate intricate biological structures and diverse functions with engineered materials. Controlling proteins at bio-solid interfaces relies on establishing key correlations between their primary sequences and resulting spatial organizations on substrates. Protein self-assembly, however, remains an engineering challenge. As a novel approach, we demonstrate here that short dodecapeptides selected by phage display are capable of self-assembly on graphite and form long-range ordered biomolecular nanostructures. Using atomic force microscopy and contact angle studies, we identify three amino-acid domains along the primary sequence that steer peptide ordering and lead to nanostructures with uniformly displayed residues. The peptides are further engineered via simple mutations to control fundamental interfacial processes, including initial binding, surface aggregation and growth kinetics, and intermolecular interactions. Tailoring short peptides via their primary sequence offers versatile control over molecular self-assembly, resulting in well-defined surface properties essential in building engineered, chemically rich, bio-solid interfaces. PMID:22233341

Molecular selection in a unified evolutionary sequence

NASA Technical Reports Server (NTRS)

Fox, S. W.

1986-01-01

With guidance from experiments and observations that indicate internally limited phenomena, an outline of unified evolutionary sequence is inferred. Such unification is not visible for a context of random matrix and random mutation. The sequence proceeds from Big Bang through prebiotic matter, protocells, through the evolving cell via molecular and natural selection, to mind, behavior, and society.

Characterization and Amplification of Gene-Based Simple Sequence Repeat (SSR) Markers in Date Palm.

PubMed

Zhao, Yongli; Keremane, Manjunath; Prakash, Channapatna S; He, Guohao

2017-01-01

The paucity of molecular markers limits the application of genetic and genomic research in date palm (Phoenix dactylifera L.). Availability of expressed sequence tag (EST) sequences in date palm may provide a good resource for developing gene-based markers. This study characterizes a substantial fraction of transcriptome sequences containing simple sequence repeats (SSRs) from the EST sequences in date palm. The EST sequences studied are mainly homologous to those of Elaeis guineensis and Musa acuminata. A total of 911 gene-based SSR markers, characterized with functional annotations, have provided a useful basis not only for discovering candidate genes and understanding genetic basis of traits of interest but also for developing genetic and genomic tools for molecular research in date palm, such as diversity study, quantitative trait locus (QTL) mapping, and molecular breeding. The procedures of DNA extraction, polymerase chain reaction (PCR) amplification of these gene-based SSR markers, and gel electrophoresis of PCR products are described in this chapter.

In silico characterization and analysis of RTBP1 and NgTRF1 protein through MD simulation and molecular docking - A comparative study.

PubMed

Mukherjee, Koel; Pandey, Dev Mani; Vidyarthi, Ambarish Saran

2015-02-06

Gaining access to sequence and structure information of telomere binding proteins helps in understanding the essential biological processes involve in conserved sequence specific interaction between DNA and the proteins. Rice telomere binding protein (RTBP1) and Nicotiana glutinosa telomere repeat binding factor (NgTRF1) are helix turn helix motif type of proteins that plays role in telomeric DNA protection and length regulation. Both the proteins share same type of domain but till now there is very less communication on the in silico studies of these complete proteins.Here we intend to do a comparative study between two proteins through modeling of the complete proteins, physiochemical characterization, MD simulation and DNA-protein docking. I-TASSER and CLC protein work bench was performed to find out the protein 3D structure as well as the different parameters to characterize the proteins. MD simulation was completed by GROMOS forcefield of GROMACS for 10 ns of time stretch. The simulated 3D structures were docked with template DNA (3D DNA modeled through 3D-DART) of TTTAGGG conserved sequence motif using HADDOCK web server.Digging up all the facts about the proteins it was reveled that around 120 amino acids in the tail part was showing a good sequence similarity between the proteins. Molecular modeling, sequence characterization and secondary structure prediction also indicates the similarity between the protein's structure and sequence. The result of MD simulation highlights on the RMSD, RMSF, Rg, PCA and Energy plots which also conveys the similar type of motional behavior between them. The best complex formation for both the proteins in docking result also indicates for the first interaction site which is mainly the helix3 region of the DNA binding domain. The overall computational analysis reveals that RTBP1 and NgTRF1 proteins display good amount of similarity in their physicochemical properties, structure, dynamics and binding mode.

In Silico Characterization and Analysis of RTBP1 and NgTRF1 Protein Through MD Simulation and Molecular Docking: A Comparative Study.

PubMed

Mukherjee, Koel; Pandey, Dev Mani; Vidyarthi, Ambarish Saran

2015-09-01

Gaining access to sequence and structure information of telomere-binding proteins helps in understanding the essential biological processes involve in conserved sequence-specific interaction between DNA and the proteins. Rice telomere-binding protein (RTBP1) and Nicotiana glutinosa telomere repeat binding factor (NgTRF1) are helix-turn-helix motif type of proteins that plays role in telomeric DNA protection and length regulation. Both the proteins share same type of domain, but till now there is very less communication on the in silico studies of these complete proteins. Here we intend to do a comparative study between two proteins through modeling of the complete proteins, physiochemical characterization, MD simulation and DNA-protein docking. I-TASSER and CLC protein work bench was performed to find out the protein 3D structure as well as the different parameters to characterize the proteins. MD simulation was completed by GROMOS forcefield of GROMACS for 10 ns of time stretch. The simulated 3D structures were docked with template DNA (3D DNA modeled through 3D-DART) of TTTAGGG conserved sequence motif using HADDOCK Web server. By digging up all the facts about the proteins, it was revealed that around 120 amino acids in the tail part were showing a good sequence similarity between the proteins. Molecular modeling, sequence characterization and secondary structure prediction also indicate the similarity between the protein's structure and sequence. The result of MD simulation highlights on the RMSD, RMSF, Rg, PCA and energy plots which also conveys the similar type of motional behavior between them. The best complex formation for both the proteins in docking result also indicates for the first interaction site which is mainly the helix3 region of the DNA-binding domain. The overall computational analysis reveals that RTBP1 and NgTRF1 proteins display good amount of similarity in their physicochemical properties, structure, dynamics and binding mode.

Modulation of Quorum Sensing in a Gram-Positive Pathogen by Linear Molecularly Imprinted Polymers with Anti-infective Properties.

PubMed

Motib, Anfal; Guerreiro, Antonio; Al-Bayati, Firas; Piletska, Elena; Manzoor, Irfan; Shafeeq, Sulman; Kadam, Anagha; Kuipers, Oscar; Hiller, Luisa; Cowen, Todd; Piletsky, Sergey; Andrew, Peter W; Yesilkaya, Hasan

2017-12-22

We describe the development, characterization, and biological testing of a new type of linear molecularly imprinted polymer (LMIP) designed to act as an anti-infective by blocking the quorum sensing (QS) mechanism and so abrogating the virulence of the pathogen Streptococcus pneumoniae. The LMIP is prepared (polymerized) in presence of a template molecule, but unlike in traditional molecular imprinting approaches, no cross-linker is used. This results in soluble low-molecular-weight oligomers that can act as a therapeutic agent in vitro and in vivo. The LMIP was characterized by mass spectrometry to determine its monomer composition. Fragments identified were then aligned along the peptide template by computer modeling to predict the possible monomer sequence of the LMIP. These findings provide a proof of principle that LMIPs can be used to block QS, thus setting the stage for the development of LMIPs a novel drug-discovery platform and class of materials to target Gram-positive pathogens. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

[Algorithm of toxigenic genetically altered Vibrio cholerae El Tor biovar strain identification].

PubMed

Smirnova, N I; Agafonov, D A; Zadnova, S P; Cherkasov, A V; Kutyrev, V V

2014-01-01

Development of an algorithm of genetically altered Vibrio cholerae biovar El Tor strai identification that ensures determination of serogroup, serovar and biovar of the studied isolate based on pheno- and genotypic properties, detection of genetically altered cholera El Tor causative agents, their differentiation by epidemic potential as well as evaluation of variability of key pathogenicity genes. Complex analysis of 28 natural V. cholerae strains was carried out by using traditional microbiological methods, PCR and fragmentary sequencing. An algorithm of toxigenic genetically altered V. cholerae biovar El Tor strain identification was developed that includes 4 stages: determination of serogroup, serovar and biovar based on phenotypic properties, confirmation of serogroup and biovar based on molecular-genetic properties determination of strains as genetically altered, differentiation of genetically altered strains by their epidemic potential and detection of ctxB and tcpA key pathogenicity gene polymorphism. The algorithm is based on the use of traditional microbiological methods, PCR and sequencing of gene fragments. The use of the developed algorithm will increase the effectiveness of detection of genetically altered variants of the cholera El Tor causative agent, their differentiation by epidemic potential and will ensure establishment of polymorphism of genes that code key pathogenicity factors for determination of origins of the strains and possible routes of introduction of the infection.

Investigating the molecular underpinnings underlying morphology and changes in carbon partitioning during tension wood formation in Eucalyptus.

PubMed

Mizrachi, Eshchar; Maloney, Victoria J; Silberbauer, Janine; Hefer, Charles A; Berger, Dave K; Mansfield, Shawn D; Myburg, Alexander A

2015-06-01

Tension wood has distinct physical and chemical properties, including altered fibre properties, cell wall composition and ultrastructure. It serves as a good system for investigating the genetic regulation of secondary cell wall biosynthesis and wood formation. The reference genome sequence for Eucalyptus grandis allows investigation of the global transcriptional reprogramming that accompanies tension wood formation in this global wood fibre crop. We report the first comprehensive analysis of physicochemical wood property changes in tension wood of Eucalyptus measured in a hybrid (E. grandis × Eucalyptus urophylla) clone, as well as genome-wide gene expression changes in xylem tissues 3 wk post-induction using RNA sequencing. We found that Eucalyptus tension wood in field-grown trees is characterized by an increase in cellulose, a reduction in lignin, xylose and mannose, and a marked increase in galactose. Gene expression profiling in tension wood-forming tissue showed corresponding down-regulation of monolignol biosynthetic genes, and differential expression of several carbohydrate active enzymes. We conclude that alterations of cell wall traits induced by tension wood formation in Eucalyptus are a consequence of a combination of down-regulation of lignin biosynthesis and hemicellulose remodelling, rather than the often proposed up-regulation of the cellulose biosynthetic pathway. © 2014 University of Pretoria New Phytologist © 2014 New Phytologist Trust.

[Sequencing and analysis of complete genome of rabies viruses isolated from Chinese Ferret-Badger and dog in Zhejiang province].

PubMed

Lei, Yong-Liang; Wang, Xiao-Guang; Tao, Xiao-Yan; Li, Hao; Meng, Sheng-Li; Chen, Xiu-Ying; Liu, Fu-Ming; Ye, Bi-Feng; Tang, Qing

2010-01-01

Based on sequencing the full-length genomes of four Chinese Ferret-Badger and dog, we analyze the properties of rabies viruses genetic variation in molecular level, get the information about rabies viruses prevalence and variation in Zhejiang, and enrich the genome database of rabies viruses street strains isolated from China. Rabies viruses in suckling mice were isolated, overlapped fragments were amplified by RT-PCR and full-length genomes were assembled to analyze the nucleotide and deduced protein similarities and phylogenetic analyses from Chinese Ferret-Badger, dog, sika deer, vole, used vaccine strain were determined. The four full-length genomes were sequenced completely and had the same genetic structure with the length of 11, 923 nts or 11, 925 nts including 58 nts-Leader, 1353 nts-NP, 894 nts-PP, 609 nts-MP, 1575 nts-GP, 6386 nts-LP, and 2, 5, 5 nts- intergenic regions(IGRs), 423 nts-Pseudogene-like sequence (psi), 70 nts-Trailer. The four full-length genomes were in accordance with the properties of Rhabdoviridae Lyssa virus by BLAST and multi-sequence alignment. The nucleotide and amino acid sequences among Chinese strains had the highest similarity, especially among animals of the same species. Of the four full-length genomes, the similarity in amino acid level was dramatically higher than that in nucleotide level, so the nucleotide mutations happened in these four genomes were most synonymous mutations. Compared with the reference rabies viruses, the lengths of the five protein coding regions had no change, no recombination, only with a few point mutations. It was evident that the five proteins appeared to be stable. The variation sites and types of the four genomes were similar to the reference vaccine or street strains. And the four strains were genotype 1 according to the multi-sequence and phylogenetic analyses, which possessed the distinct district characteristics of China. Therefore, these four rabies viruses are likely to be street viruses already existing in the natural world.

Characterization of Polyhydroxyalkanoate Produced by Bacillus megaterium VB89 Isolated from Nisargruna Biogas Plant.

PubMed

Baikar, Vishakha; Rane, Ashwini; Deopurkar, Rajendra

2017-09-01

Polyhydroxyalkanoates (PHAs) are naturally occurring biodegradable polymers that can curb the extensive use of polypropylene based plastics. In contrast to chemically synthesized polypropylene plastics, PHAs are biodegradable and thus environmentally safe. PHAs have attracted much attention as biocompatible and biodegradable thermoplastics. The present study involves isolation of bacteria from different environments capable of synthesizing PHAs. The bacterium producing highest yield of PHA (0.672 ± 0.041 g/L) was identified as Bacillus megaterium VB89 by biochemical and molecular techniques such as 16S rDNA sequence analysis. Strain VB89 produced polyhydroxybutyrate (PHB) as revealed by FTIR and NMR. This PHB had an average molecular weight of 2.89 × 10 5  Da and a polydispersity index of 2.37. Thermal properties of the PHB included a glass transition temperature of 13.97 °C, a melting temperature of 181.74 °C, and a decomposition temperature of 234 °C. All these properties indicated that VB89 produced PHB of high purity and good thermal stability.

Genetic Code Expansion as a Tool to Study Regulatory Processes of Transcription

NASA Astrophysics Data System (ADS)

Schmidt, Moritz; Summerer, Daniel

2014-02-01

The expansion of the genetic code with noncanonical amino acids (ncAA) enables the chemical and biophysical properties of proteins to be tailored, inside cells, with a previously unattainable level of precision. A wide range of ncAA with functions not found in canonical amino acids have been genetically encoded in recent years and have delivered insights into biological processes that would be difficult to access with traditional approaches of molecular biology. A major field for the development and application of novel ncAA-functions has been transcription and its regulation. This is particularly attractive, since advanced DNA sequencing- and proteomics-techniques continue to deliver vast information on these processes on a global level, but complementing methodologies to study them on a detailed, molecular level and in living cells have been comparably scarce. In a growing number of studies, genetic code expansion has now been applied to precisely control the chemical properties of transcription factors, RNA polymerases and histones, and this has enabled new insights into their interactions, conformational changes, cellular localizations and the functional roles of posttranslational modifications.

Microbial mat of the thermal springs Kuchiger Republic of Buryatia: species composition, biochemical properties and electrogenic activity in biofuel cell

NASA Astrophysics Data System (ADS)

Aleksandrovich Yuriev, Denis; Viktorovna Zaitseva, Svetlana; Olegovna Zhdanova, Galina; Yurievich Tolstoy, Mikhail; Dondokovna Barkhutova, Darima; Feodorovna Vyatchina, Olga; Yuryevna Konovalova, Elena; Iosifovich Stom, Devard

2018-02-01

Electrogenic, molecular and some other properties of a microbial mat isolated from the Kuchiger hot spring (Kurumkansky District, Republic of Buryatia) were studied. Molecular analysis showed that representatives of Proteobacteria (85.5 % of the number of classified bacterial sequences) prevailed in the microbial mat of the Kuchiger springs, among which sulfur bacteria of the genus Thiothrix were the most numerous. In the microbial mat there were bacteria from the families Rhodocyclaceae, Comamonadaceae and Flavobacteriaceae. Phylum Bacteroidetes, Cyanobacteria/Chloroplast, Fusobacteria, Fibrobacteres, Acidobacteria, Chlorobi, Spirochaetes, Verrucomicrobia, Firmicutes, Deinococcus-Thermus, Chloroflexi and Actinobacteria are also noted in the composition of the microbial mat. Under the experimental conditions using Kuchiger-mat 16 as bioagents, glucose and peptone as substrates, the power of BFC was 240 and 221 mW / m2, respectively. When replacing the substrate with sodium acetate, the efficiency of the BFC was reduced by a factor of 10 (20 mW / m2). The prospects of using a microbial mat “Kuchiger-16” as an electrogen in BFC when utilizing alkaline waste water components to generate electricity are discussed.

Chemically specific coarse-grained models to investigate the structure of biomimetic membranes

DOE PAGES

Kowalik, Ma?gorzata; Schantz, Allen B.; Naqi, Abdullah; ...

2017-11-29

Biomimetic polymer/protein membranes are promising materials for DNA sequencing, sensors, drug delivery and water purification. These self-assembled structures are made from low molecular weight amphiphilic block copolymers (N hydrophobic < 40 for a diblock copolymer), including poly(ethylene oxide)–1,2-polybutadiene (EO–1,2-BD) and poly(ethylene oxide)–poly(ethyl ethylene) (EO–EE). To examine these membranes' nanoscale structure, we developed a coarse-grained molecular dynamics (CG MD) model for EO–1,2-BD and assembled a CG MD model for EO–EE using parameters from two published force fields. We observe that the polymers' hydrophobic core blocks are slightly stretched compared to the random coil configuration seen at higher molecular weights. We alsomore » observe an increase in the interdigitation of the hydrophobic leaflets with increasing molecular weight (consistent with literature). The hydration level of the EO corona (which may influence protein incorporation) is higher for membranes with a larger area/chain, regardless of whether EE or 1,2-BD forms the hydrophobic block. Our results provide a molecular-scale view of membrane packing and hydrophobicity, two important properties for creating polymer–protein biomimetic membranes.« less

Chemically specific coarse-grained models to investigate the structure of biomimetic membranes

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

Kowalik, Ma?gorzata; Schantz, Allen B.; Naqi, Abdullah

Biomimetic polymer/protein membranes are promising materials for DNA sequencing, sensors, drug delivery and water purification. These self-assembled structures are made from low molecular weight amphiphilic block copolymers (N hydrophobic < 40 for a diblock copolymer), including poly(ethylene oxide)–1,2-polybutadiene (EO–1,2-BD) and poly(ethylene oxide)–poly(ethyl ethylene) (EO–EE). To examine these membranes' nanoscale structure, we developed a coarse-grained molecular dynamics (CG MD) model for EO–1,2-BD and assembled a CG MD model for EO–EE using parameters from two published force fields. We observe that the polymers' hydrophobic core blocks are slightly stretched compared to the random coil configuration seen at higher molecular weights. We alsomore » observe an increase in the interdigitation of the hydrophobic leaflets with increasing molecular weight (consistent with literature). The hydration level of the EO corona (which may influence protein incorporation) is higher for membranes with a larger area/chain, regardless of whether EE or 1,2-BD forms the hydrophobic block. Our results provide a molecular-scale view of membrane packing and hydrophobicity, two important properties for creating polymer–protein biomimetic membranes.« less

Molecular evolution of the polyamine oxidase gene family in Metazoa

PubMed Central

2012-01-01

Background Polyamine oxidase enzymes catalyze the oxidation of polyamines and acetylpolyamines. Since polyamines are basic regulators of cell growth and proliferation, their homeostasis is crucial for cell life. Members of the polyamine oxidase gene family have been identified in a wide variety of animals, including vertebrates, arthropodes, nematodes, placozoa, as well as in plants and fungi. Polyamine oxidases (PAOs) from yeast can oxidize spermine, N1-acetylspermine, and N1-acetylspermidine, however, in vertebrates two different enzymes, namely spermine oxidase (SMO) and acetylpolyamine oxidase (APAO), specifically catalyze the oxidation of spermine, and N1-acetylspermine/N1-acetylspermidine, respectively. Little is known about the molecular evolutionary history of these enzymes. However, since the yeast PAO is able to catalyze the oxidation of both acetylated and non acetylated polyamines, and in vertebrates these functions are addressed by two specialized polyamine oxidase subfamilies (APAO and SMO), it can be hypothesized an ancestral reference for the former enzyme from which the latter would have been derived. Results We analysed 36 SMO, 26 APAO, and 14 PAO homologue protein sequences from 54 taxa including various vertebrates and invertebrates. The analysis of the full-length sequences and the principal domains of vertebrate and invertebrate PAOs yielded consensus primary protein sequences for vertebrate SMOs and APAOs, and invertebrate PAOs. This analysis, coupled to molecular modeling techniques, also unveiled sequence regions that confer specific structural and functional properties, including substrate specificity, by the different PAO subfamilies. Molecular phylogenetic trees revealed a basal position of all the invertebrates PAO enzymes relative to vertebrate SMOs and APAOs. PAOs from insects constitute a monophyletic clade. Two PAO variants sampled in the amphioxus are basal to the dichotomy between two well supported monophyletic clades including, respectively, all the SMOs and APAOs from vertebrates. The two vertebrate monophyletic clades clustered strictly mirroring the organismal phylogeny of fishes, amphibians, reptiles, birds, and mammals. Evidences from comparative genomic analysis, structural evolution and functional divergence in a phylogenetic framework across Metazoa suggested an evolutionary scenario where the ancestor PAO coding sequence, present in invertebrates as an orthologous gene, has been duplicated in the vertebrate branch to originate the paralogous SMO and APAO genes. A further genome evolution event concerns the SMO gene of placental, but not marsupial and monotremate, mammals which increased its functional variation following an alternative splicing (AS) mechanism. Conclusions In this study the explicit integration in a phylogenomic framework of phylogenetic tree construction, structure prediction, and biochemical function data/prediction, allowed inferring the molecular evolutionary history of the PAO gene family and to disambiguate paralogous genes related by duplication event (SMO and APAO) and orthologous genes related by speciation events (PAOs, SMOs/APAOs). Further, while in vertebrates experimental data corroborate SMO and APAO molecular function predictions, in invertebrates the finding of a supported phylogenetic clusters of insect PAOs and the co-occurrence of two PAO variants in the amphioxus urgently claim the need for future structure-function studies. PMID:22716069

Molecular evolution of the polyamine oxidase gene family in Metazoa.

PubMed

Polticelli, Fabio; Salvi, Daniele; Mariottini, Paolo; Amendola, Roberto; Cervelli, Manuela

2012-06-20

Polyamine oxidase enzymes catalyze the oxidation of polyamines and acetylpolyamines. Since polyamines are basic regulators of cell growth and proliferation, their homeostasis is crucial for cell life. Members of the polyamine oxidase gene family have been identified in a wide variety of animals, including vertebrates, arthropodes, nematodes, placozoa, as well as in plants and fungi. Polyamine oxidases (PAOs) from yeast can oxidize spermine, N1-acetylspermine, and N1-acetylspermidine, however, in vertebrates two different enzymes, namely spermine oxidase (SMO) and acetylpolyamine oxidase (APAO), specifically catalyze the oxidation of spermine, and N1-acetylspermine/N1-acetylspermidine, respectively. Little is known about the molecular evolutionary history of these enzymes. However, since the yeast PAO is able to catalyze the oxidation of both acetylated and non acetylated polyamines, and in vertebrates these functions are addressed by two specialized polyamine oxidase subfamilies (APAO and SMO), it can be hypothesized an ancestral reference for the former enzyme from which the latter would have been derived. We analysed 36 SMO, 26 APAO, and 14 PAO homologue protein sequences from 54 taxa including various vertebrates and invertebrates. The analysis of the full-length sequences and the principal domains of vertebrate and invertebrate PAOs yielded consensus primary protein sequences for vertebrate SMOs and APAOs, and invertebrate PAOs. This analysis, coupled to molecular modeling techniques, also unveiled sequence regions that confer specific structural and functional properties, including substrate specificity, by the different PAO subfamilies. Molecular phylogenetic trees revealed a basal position of all the invertebrates PAO enzymes relative to vertebrate SMOs and APAOs. PAOs from insects constitute a monophyletic clade. Two PAO variants sampled in the amphioxus are basal to the dichotomy between two well supported monophyletic clades including, respectively, all the SMOs and APAOs from vertebrates. The two vertebrate monophyletic clades clustered strictly mirroring the organismal phylogeny of fishes, amphibians, reptiles, birds, and mammals. Evidences from comparative genomic analysis, structural evolution and functional divergence in a phylogenetic framework across Metazoa suggested an evolutionary scenario where the ancestor PAO coding sequence, present in invertebrates as an orthologous gene, has been duplicated in the vertebrate branch to originate the paralogous SMO and APAO genes. A further genome evolution event concerns the SMO gene of placental, but not marsupial and monotremate, mammals which increased its functional variation following an alternative splicing (AS) mechanism. In this study the explicit integration in a phylogenomic framework of phylogenetic tree construction, structure prediction, and biochemical function data/prediction, allowed inferring the molecular evolutionary history of the PAO gene family and to disambiguate paralogous genes related by duplication event (SMO and APAO) and orthologous genes related by speciation events (PAOs, SMOs/APAOs). Further, while in vertebrates experimental data corroborate SMO and APAO molecular function predictions, in invertebrates the finding of a supported phylogenetic clusters of insect PAOs and the co-occurrence of two PAO variants in the amphioxus urgently claim the need for future structure-function studies.

Deciphering the molecular mechanisms underlying the binding of the TWIST1/E12 complex to regulatory E-box sequences

PubMed Central

Bouard, Charlotte; Terreux, Raphael; Honorat, Mylène; Manship, Brigitte; Ansieau, Stéphane; Vigneron, Arnaud M.; Puisieux, Alain; Payen, Léa

2016-01-01

Abstract The TWIST1 bHLH transcription factor controls embryonic development and cancer processes. Although molecular and genetic analyses have provided a wealth of data on the role of bHLH transcription factors, very little is known on the molecular mechanisms underlying their binding affinity to the E-box sequence of the promoter. Here, we used an in silico model of the TWIST1/E12 (TE) heterocomplex and performed molecular dynamics (MD) simulations of its binding to specific (TE-box) and modified E-box sequences. We focused on (i) active E-box and inactive E-box sequences, on (ii) modified active E-box sequences, as well as on (iii) two box sequences with modified adjacent bases the AT- and TA-boxes. Our in silico models were supported by functional in vitro binding assays. This exploration highlighted the predominant role of protein side-chain residues, close to the heart of the complex, at anchoring the dimer to DNA sequences, and unveiled a shift towards adjacent ((-1) and (-1*)) bases and conserved bases of modified E-box sequences. In conclusion, our study provides proof of the predictive value of these MD simulations, which may contribute to the characterization of specific inhibitors by docking approaches, and their use in pharmacological therapies by blocking the tumoral TWIST1/E12 function in cancers. PMID:27151200

Serological Analysis of Immunogenic Properties of Recombinant Meningococcus IgA1 Protease-Based Proteins.

PubMed

Kotelnikova, O V; Zinchenko, A A; Vikhrov, A A; Alliluev, A P; Serova, O V; Gordeeva, E A; Zhigis, L S; Zueva, V S; Razgulyaeva, O A; Melikhova, T D; Nokel, E A; Drozhzhina, E Yu; Rumsh, L D

2016-07-01

Using the genome sequence of IgA1 protease of N. meningitidis of serogroup B, four recombinant proteins of different structure and molecular weight were constructed. These proteins were equal in inducing the formation of specific antibodies to IgA1 protease and had protective properties against meningococci. In the sera of immunized mice, anti-IgA1 protease antibodies were detected by whole-cell ELISA, which indicated the presence of IgA1 protease on the surface of these bacteria. We hypothesized that the protective properties of IgA1 protease-based antigens and IgA1 protease analogs could be realized not only via impairment of bacterium adhesion to the mucosa, but also via suppression of this pathogen in the organism. The presented findings seem promising for using these proteins as the basis for anti-meningococcus vaccine.

Focus on PNA Flexibility and RNA Binding using Molecular Dynamics and Metadynamics

NASA Astrophysics Data System (ADS)

Verona, Massimiliano Donato; Verdolino, Vincenzo; Palazzesi, Ferruccio; Corradini, Roberto

2017-02-01

Peptide Nucleic Acids (PNAs) can efficiently target DNA or RNA acting as chemical tools for gene regulation. Their backbone modification and functionalization is often used to increase the affinity for a particular sequence improving selectivity. The understanding of the trading forces that lead the single strand PNA to bind the DNA or RNA sequence is preparatory for any further rational design, but a clear and unique description of this process is still not complete. In this paper we report further insights into this subject, by a computational investigation aiming at the characterization of the conformations of a single strand PNA and how these can be correlated to its capability in binding DNA/RNA. Employing Metadynamics we were able to better define conformational pre-organizations of the single strand PNA and γ-modified PNA otherwise unrevealed through classical molecular dynamics. Our simulations driven on backbone modified PNAs lead to the conclusion that this γ-functionalization affects the single strand preorganization and targeting properties to the DNA/RNA, in agreement with circular dichroism (CD) spectra obtained for this class of compounds. MD simulations on PNA:RNA dissociation and association mechanisms allowed to reveal the critical role of central bases and preorganization in the binding process.

Transcriptome Analysis of Differentially Expressed Genes Provides Insight into Stolon Formation in Tulipa edulis

PubMed Central

Miao, Yuanyuan; Zhu, Zaibiao; Guo, Qiaosheng; Zhu, Yunhao; Yang, Xiaohua; Sun, Yuan

2016-01-01

Tulipa edulis (Miq.) Baker is an important medicinal plant with a variety of anti-cancer properties. The stolon is one of the main asexual reproductive organs of T. edulis and possesses a unique morphology. To explore the molecular mechanism of stolon formation, we performed an RNA-seq analysis of the transcriptomes of stolons at three developmental stages. In the present study, 15.49 Gb of raw data were generated and assembled into 74,006 unigenes, and a total of 2,811 simple sequence repeats were detected in T. edulis. Among the three libraries of stolons at different developmental stages, there were 5,119 differentially expressed genes (DEGs). A functional annotation analysis based on sequence similarity queries of the GO, COG, KEGG databases showed that these DEGs were mainly involved in many physiological and biochemical processes, such as material and energy metabolism, hormone signaling, cell growth, and transcription regulation. In addition, quantitative real-time PCR analysis revealed that the expression patterns of the DEGs were consistent with the transcriptome data, which further supported a role for the DEGs in stolon formation. This study provides novel resources for future genetic and molecular studies in T. edulis. PMID:27064558

Transcriptome Analysis of Differentially Expressed Genes Provides Insight into Stolon Formation in Tulipa edulis.

PubMed

Miao, Yuanyuan; Zhu, Zaibiao; Guo, Qiaosheng; Zhu, Yunhao; Yang, Xiaohua; Sun, Yuan

2016-01-01

Tulipa edulis (Miq.) Baker is an important medicinal plant with a variety of anti-cancer properties. The stolon is one of the main asexual reproductive organs of T. edulis and possesses a unique morphology. To explore the molecular mechanism of stolon formation, we performed an RNA-seq analysis of the transcriptomes of stolons at three developmental stages. In the present study, 15.49 Gb of raw data were generated and assembled into 74,006 unigenes, and a total of 2,811 simple sequence repeats were detected in T. edulis. Among the three libraries of stolons at different developmental stages, there were 5,119 differentially expressed genes (DEGs). A functional annotation analysis based on sequence similarity queries of the GO, COG, KEGG databases showed that these DEGs were mainly involved in many physiological and biochemical processes, such as material and energy metabolism, hormone signaling, cell growth, and transcription regulation. In addition, quantitative real-time PCR analysis revealed that the expression patterns of the DEGs were consistent with the transcriptome data, which further supported a role for the DEGs in stolon formation. This study provides novel resources for future genetic and molecular studies in T. edulis.

Focus on PNA Flexibility and RNA Binding using Molecular Dynamics and Metadynamics.

PubMed

Verona, Massimiliano Donato; Verdolino, Vincenzo; Palazzesi, Ferruccio; Corradini, Roberto

2017-02-17

Peptide Nucleic Acids (PNAs) can efficiently target DNA or RNA acting as chemical tools for gene regulation. Their backbone modification and functionalization is often used to increase the affinity for a particular sequence improving selectivity. The understanding of the trading forces that lead the single strand PNA to bind the DNA or RNA sequence is preparatory for any further rational design, but a clear and unique description of this process is still not complete. In this paper we report further insights into this subject, by a computational investigation aiming at the characterization of the conformations of a single strand PNA and how these can be correlated to its capability in binding DNA/RNA. Employing Metadynamics we were able to better define conformational pre-organizations of the single strand PNA and γ-modified PNA otherwise unrevealed through classical molecular dynamics. Our simulations driven on backbone modified PNAs lead to the conclusion that this γ-functionalization affects the single strand preorganization and targeting properties to the DNA/RNA, in agreement with circular dichroism (CD) spectra obtained for this class of compounds. MD simulations on PNA:RNA dissociation and association mechanisms allowed to reveal the critical role of central bases and preorganization in the binding process.

A Novel High-Molecular-Mass Bacteriocin Produced by Enterococcus faecium: Biochemical Features and Mode of Action.

PubMed

Vasilchenko, A S; Vasilchenko, A V; Valyshev, A V; Rogozhin, E A

2018-02-08

Discovery of a novel bacteriocin is always an event in sciences, since cultivation of most bacterial species is a general problem in microbiology. This statement is reflected by the fact that number of bacteriocins is smaller for tenfold comparing to known antimicrobial peptides. We cultivated Enterococcus faecium on simplified medium to reduce amount of purification steps. This approach allows to purify the novel heavy weight bacteriocin produced by E. faecium ICIS 7. The novelty of this bacteriocin, named enterocin-7, was confirmed by N-terminal sequencing and by comparing the structural-functional properties with available data. Purified enterocin-7 is characterized by a sequence of amino acid residues having no homology in UniProt/SwissProt/TrEMBL databases: NH2 - Asp - Ala - His - Leu - Ser - Glu - Val - Ala - Glu - Arg - Phe - Glu - Asp - Leu - Gly. Isolated thermostable protein has a molecular mass of 65 kDa, which allows it to be classified into class III in bacteriocin classification schemes. Enterocin-7 displayed a broad spectrum of activity against some Gram-positive and Gram-negative microorganisms. Fluorescent microscopy and spectroscopy showed the permeabilizing mechanism of the action of enterocin-7, which is realized within a few minutes.

Membrane insertion of fusion peptides from Ebola and Marburg viruses studied by replica-exchange molecular dynamics simulations.

PubMed

Olson, Mark A; Lee, Michael S; Yeh, In-Chul

2017-06-15

This work presents replica-exchange molecular dynamics simulations of inserting a 16-residue Ebola virus fusion peptide into a membrane bilayer. A computational approach is applied for modeling the peptide at the explicit all-atom level and the membrane-aqueous bilayer by a generalized Born continuum model with a smoothed switching function (GBSW). We provide an assessment of the model calculations in terms of three metrics: (1) the ability to reproduce the NMR structure of the peptide determined in the presence of SDS micelles and comparable structural data on other fusion peptides; (2) determination of the effects of the mutation Trp-8 to Ala and sequence discrimination of the homologous Marburg virus; and (3) calculation of potentials of mean force for estimating the partitioning free energy and their comparison to predictions from the Wimley-White interfacial hydrophobicity scale. We found the GBSW implicit membrane model to produce results of limited accuracy in conformational properties of the peptide when compared to the NMR structure, yet the model resolution is sufficient to determine the effect of sequence differentiation on peptide-membrane integration. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

RNAmutants: a web server to explore the mutational landscape of RNA secondary structures

PubMed Central

Waldispühl, Jerome; Devadas, Srinivas; Berger, Bonnie; Clote, Peter

2009-01-01

The history and mechanism of molecular evolution in DNA have been greatly elucidated by contributions from genetics, probability theory and bioinformatics—indeed, mathematical developments such as Kimura's neutral theory, Kingman's coalescent theory and efficient software such as BLAST, ClustalW, Phylip, etc., provide the foundation for modern population genetics. In contrast to DNA, the function of most noncoding RNA depends on tertiary structure, experimentally known to be largely determined by secondary structure, for which dynamic programming can efficiently compute the minimum free energy secondary structure. For this reason, understanding the effect of pointwise mutations in RNA secondary structure could reveal fundamental properties of structural RNA molecules and improve our understanding of molecular evolution of RNA. The web server RNAmutants provides several efficient tools to compute the ensemble of low-energy secondary structures for all k-mutants of a given RNA sequence, where k is bounded by a user-specified upper bound. As we have previously shown, these tools can be used to predict putative deleterious mutations and to analyze regulatory sequences from the hepatitis C and human immunodeficiency genomes. Web server is available at http://bioinformatics.bc.edu/clotelab/RNAmutants/, and downloadable binaries at http://rnamutants.csail.mit.edu/. PMID:19531740

Stepwise Mechanism of Temperature-Dependent Coacervation of the Elastin-like Peptide Analogue Dimer, (C(WPGVG)3)2.

PubMed

Tatsubo, Daiki; Suyama, Keitaro; Miyazaki, Masaya; Maeda, Iori; Nose, Takeru

2018-03-13

Elastin-like peptides (ELPs) are distinct, repetitive, hydrophobic sequences, such as (VPGVG) n , that exhibit coacervation, the property of reversible, temperature-dependent self-association and dissociation. ELPs can be found in elastin and have been developed as new scaffold biomaterials. However, the detailed relationship between their amino acid sequences and coacervation properties remains obscure because of the structural flexibility of ELPs. In this study, we synthesized a novel, dimeric ELP analogue (H-C(WPGVG) 3 -NH 2 ) 2 , henceforth abbreviated (CW3)2, and analyzed its self-assembly properties and structural factors as indicators of coacervation. Turbidity measurements showed that (CW3)2 demonstrated coacervation at a concentration much lower than that of its monomeric form and another ELP. In addition, the coacervate held water-soluble dye molecules. Thus, potent and distinct coacervation was obtained with a remarkably short sequence of (CW3)2. Furthermore, fluorescence microscopy, dynamic light scattering, and optical microscopy revealed that the coacervation of (CW3)2 was a stepwise process. The structural factors of (CW3)2 were analyzed by molecular dynamics simulations and circular dichroism spectroscopy. These measurements indicated that helical structures primarily consisting of proline and glycine became more disordered at high temperatures with concurrent, significant exposure of their hydrophobic surfaces. This extreme change in the hydrophobic surface contributes to the potent coacervation observed for (CW3)2. These results provide important insights into more efficient applications of ELPs and their analogues, as well as the coacervation mechanisms of ELP and elastin.

Free Energy Perturbation Calculations of the Thermodynamics of Protein Side-Chain Mutations.

PubMed

Steinbrecher, Thomas; Abel, Robert; Clark, Anthony; Friesner, Richard

2017-04-07

Protein side-chain mutation is fundamental both to natural evolutionary processes and to the engineering of protein therapeutics, which constitute an increasing fraction of important medications. Molecular simulation enables the prediction of the effects of mutation on properties such as binding affinity, secondary and tertiary structure, conformational dynamics, and thermal stability. A number of widely differing approaches have been applied to these predictions, including sequence-based algorithms, knowledge-based potential functions, and all-atom molecular mechanics calculations. Free energy perturbation theory, employing all-atom and explicit-solvent molecular dynamics simulations, is a rigorous physics-based approach for calculating thermodynamic effects of, for example, protein side-chain mutations. Over the past several years, we have initiated an investigation of the ability of our most recent free energy perturbation methodology to model the thermodynamics of protein mutation for two specific problems: protein-protein binding affinities and protein thermal stability. We highlight recent advances in the field and outline current and future challenges. Copyright © 2017 Elsevier Ltd. All rights reserved.

Molecular dynamics simulation of a needle-sphere binary mixture

NASA Astrophysics Data System (ADS)

Raghavan, Karthik

This paper investigates the dynamic behaviour of a hard needle-sphere binary system using a novel numerical technique called the Newton homotopy continuation (NHC) method. This mixture is representative of a polymer melt where both long chain molecules and monomers coexist. Since the intermolecular forces are generated from hard body interactions, the consequence of missed collisions or incorrect collision sequences have a significant bearing on the dynamic properties of the fluid. To overcome this problem, in earlier work NHC was chosen over traditional Newton-Raphson methods to solve the hard body dynamics of a needle fluid in random media composed of overlapping spheres. Furthermore, the simplicity of interactions and dynamics allows us to focus our research directly on the effects of particle shape and density on the transport behaviour of the mixture. These studies are also compared with earlier works that examined molecular chains in porous media primarily to understand the differences in molecular transport in the bulk versus porous systems.

Aptamer-Targeted Gold Nanoparticles As Molecular-Specific Contrast Agents for Reflectance Imaging

PubMed Central

2008-01-01

Targeted metallic nanoparticles have shown potential as a platform for development of molecular-specific contrast agents. Aptamers have recently been demonstrated as ideal candidates for molecular targeting applications. In this study, we investigated the development of aptamer-based gold nanoparticles as contrast agents, using aptamers as targeting agents and gold nanoparticles as imaging agents. We devised a novel conjugation approach using an extended aptamer design where the extension is complementary to an oligonucleotide sequence attached to the surface of the gold nanoparticles. The chemical and optical properties of the aptamer−gold conjugates were characterized using size measurements and oligonucleotide quantitation assays. We demonstrate this conjugation approach to create a contrast agent designed for detection of prostate-specific membrane antigen (PSMA), obtaining reflectance images of PSMA(+) and PSMA(−) cell lines treated with the anti-PSMA aptamer−gold conjugates. This design strategy can easily be modified to incorporate multifunctional agents as part of a multimodal platform for reflectance imaging applications. PMID:18512972

Directional Darwinian Selection in proteins.

PubMed

McClellan, David A

2013-01-01

Molecular evolution is a very active field of research, with several complementary approaches, including dN/dS, HON90, MM01, and others. Each has documented strengths and weaknesses, and no one approach provides a clear picture of how natural selection works at the molecular level. The purpose of this work is to present a simple new method that uses quantitative amino acid properties to identify and characterize directional selection in proteins. Inferred amino acid replacements are viewed through the prism of a single physicochemical property to determine the amount and direction of change caused by each replacement. This allows the calculation of the probability that the mean change in the single property associated with the amino acid replacements is equal to zero (H0: μ = 0; i.e., no net change) using a simple two-tailed t-test. Example data from calanoid and cyclopoid copepod cytochrome oxidase subunit I sequence pairs are presented to demonstrate how directional selection may be linked to major shifts in adaptive zones, and that convergent evolution at the whole organism level may be the result of convergent protein adaptations. Rather than replace previous methods, this new method further complements existing methods to provide a holistic glimpse of how natural selection shapes protein structure and function over evolutionary time.

Mining and gene ontology based annotation of SSR markers from expressed sequence tags of Humulus lupulus

PubMed Central

Singh, Swati; Gupta, Sanchita; Mani, Ashutosh; Chaturvedi, Anoop

2012-01-01

Humulus lupulus is commonly known as hops, a member of the family moraceae. Currently many projects are underway leading to the accumulation of voluminous genomic and expressed sequence tag sequences in public databases. The genetically characterized domains in these databases are limited due to non-availability of reliable molecular markers. The large data of EST sequences are available in hops. The simple sequence repeat markers extracted from EST data are used as molecular markers for genetic characterization, in the present study. 25,495 EST sequences were examined and assembled to get full-length sequences. Maximum frequency distribution was shown by mononucleotide SSR motifs i.e. 60.44% in contig and 62.16% in singleton where as minimum frequency are observed for hexanucleotide SSR in contig (0.09%) and pentanucleotide SSR in singletons (0.12%). Maximum trinucleotide motifs code for Glutamic acid (GAA) while AT/TA were the most frequent repeat of dinucleotide SSRs. Flanking primer pairs were designed in-silico for the SSR containing sequences. Functional categorization of SSRs containing sequences was done through gene ontology terms like biological process, cellular component and molecular function. PMID:22368382

Molecular cloning, expression and characterization of a functional GSTmu class from the cattle tick Boophilus annulatus.

PubMed

Shahein, Yasser Ezzat; El Sayed El-Hakim, Amr; Abouelella, Amira Mohamed Kamal; Hamed, Ragaa Reda; Allam, Shaimaa Abdul-Moez; Farid, Nevin Mahmoud

2008-03-25

A full-length cDNA of a glutathione S-transferase (GST) was cloned from a cDNA library of the local Egyptian cattle tick Boophilus annulatus. The 672 bp cloned fragment was sequenced and showed an open reading frame encoding a protein of 223 amino acids. Comparison of the deduced amino acid sequence with GSTs from other species revealed that the sequence is closely related to the mammalian mu-class GST. The cloned gene was expressed in E. coli under T7 promotor of pET-30b vector, and purified under native conditions. The purified enzyme appeared as a single band on 12% SDS-PAGE and has a molecular weight of 30.8 kDa including the histidine tag of the vector. The purified enzyme was assayed upon the chromogenic substrate 1-chloro-2,4-dinitrobenzene (CDNB) and the recombinant enzyme showed high level of activity even in the presence of the beta-galactosidase region on its 5' end and showed maximum activity at pH 7.5. The Km values for CDNB and GSH were 0.57 and 0.79 mM, respectively. The over expressed rBaGST showed high activity toward CDNB (121 units/mg protein) and less toward DCNB (29.3 units/mg protein). rBaGST exhibited peroxidatic activity on cumene hydroperoxide sharing this property with GSTs belonging to the GST alpha class. I50 values for cibacron blue and bromosulfophthalein were 0.22 and 8.45 microM, respectively, sharing this property with the mammalian GSTmu class. Immunoblotting revealed the presence of the GST molecule in B. annulatus protein extracts; whole tick, larvae, gut, salivary gland and ovary. Homologues to the GSTmu were also detected in other tick species as Hyalomma dromedarii and Rhipicephalus sp. while in Ornithodoros moubata, GSTmu homologue could not be detected.

Molecular-Sized DNA or RNA Sequencing Machine | NCI Technology Transfer Center | TTC

Cancer.gov

The National Cancer Institute's Gene Regulation and Chromosome Biology Laboratory is seeking statements of capability or interest from parties interested in collaborative research to co-develop a molecular-sized DNA or RNA sequencing machine.

Opportunities and challenges associated with clinical diagnostic genome sequencing: a report of the Association for Molecular Pathology.

PubMed

Schrijver, Iris; Aziz, Nazneen; Farkas, Daniel H; Furtado, Manohar; Gonzalez, Andrea Ferreira; Greiner, Timothy C; Grody, Wayne W; Hambuch, Tina; Kalman, Lisa; Kant, Jeffrey A; Klein, Roger D; Leonard, Debra G B; Lubin, Ira M; Mao, Rong; Nagan, Narasimhan; Pratt, Victoria M; Sobel, Mark E; Voelkerding, Karl V; Gibson, Jane S

2012-11-01

This report of the Whole Genome Analysis group of the Association for Molecular Pathology illuminates the opportunities and challenges associated with clinical diagnostic genome sequencing. With the reality of clinical application of next-generation sequencing, technical aspects of molecular testing can be accomplished at greater speed and with higher volume, while much information is obtained. Although this testing is a next logical step for molecular pathology laboratories, the potential impact on the diagnostic process and clinical correlations is extraordinary and clinical interpretation will be challenging. We review the rapidly evolving technologies; provide application examples; discuss aspects of clinical utility, ethics, and consent; and address the analytic, postanalytic, and professional implications. Copyright © 2012 American Society for Investigative Pathology and the Association for Molecular Pathology. Published by Elsevier Inc. All rights reserved.

Mapping Simple Repeated DNA Sequences in Heterochromatin of Drosophila Melanogaster

PubMed Central

Lohe, A. R.; Hilliker, A. J.; Roberts, P. A.

1993-01-01

Heterochromatin in Drosophila has unusual genetic, cytological and molecular properties. Highly repeated DNA sequences (satellites) are the principal component of heterochromatin. Using probes from cloned satellites, we have constructed a chromosome map of 10 highly repeated, simple DNA sequences in heterochromatin of mitotic chromosomes of Drosophila melanogaster. Despite extensive sequence homology among some satellites, chromosomal locations could be distinguished by stringent in situ hybridizations for each satellite. Only two of the localizations previously determined using gradient-purified bulk satellite probes are correct. Eight new satellite localizations are presented, providing a megabase-level chromosome map of one-quarter of the genome. Five major satellites each exhibit a multichromosome distribution, and five minor satellites hybridize to single sites on the Y chromosome. Satellites closely related in sequence are often located near one another on the same chromosome. About 80% of Y chromosome DNA is composed of nine simple repeated sequences, in particular (AAGAC)(n) (8 Mb), (AAGAG)(n) (7 Mb) and (AATAT)(n) (6 Mb). Similarly, more than 70% of the DNA in chromosome 2 heterochromatin is composed of five simple repeated sequences. We have also generated a high resolution map of satellites in chromosome 2 heterochromatin, using a series of translocation chromosomes whose breakpoints in heterochromatin were ordered by N-banding. Finally, staining and banding patterns of heterochromatic regions are correlated with the locations of specific repeated DNA sequences. The basis for the cytochemical heterogeneity in banding appears to depend exclusively on the different satellite DNAs present in heterochromatin. PMID:8375654

Molecular confirmation of Hepatozoon canis in Mauritius.

PubMed

Daskalaki, Aikaterini Alexandra; Ionică, Angela Monica; Jeetah, Keshav; Gherman, Călin Mircea; Mihalca, Andrei Daniel

2018-01-01

In this study, Hepatozoon species was molecularly identified and characterized for the first time on the Indian Ocean island of Mauritius. Partial sequences of the 18S rRNA gene of the Hepatozoon isolates were analysed from three naturally infected dogs. The sequences of H. canis were similar to the 18S rRNA partial sequences (JX112783, AB365071 99%) from dog blood samples from West Indies and Nigeria. Our sequences were deposited in the GenBank database. Copyright © 2017 Elsevier B.V. All rights reserved.

A molecular model for illegitimate recombination in Bacillus subtilis.

PubMed

Temeyer, K B; Hopkins, K M; Chapman, L F

1991-01-01

The recombinant DNA junctions at which pUB110 and Bacillus subtilis chromosomal DNA were joined to form the plasmid pKBT1 were cloned and sequenced. From the sequencing data we conclude that the pUB110 sequence is intact in the pair of cloned pKBT1 fragments and pTL12 sequences are not present. A molecular model for the formation of pKBT1 based on structural motifs characteristic of the joint sites is presented.

A public HTLV-1 molecular epidemiology database for sequence management and data mining.

PubMed

Araujo, Thessika Hialla Almeida; Souza-Brito, Leandro Inacio; Libin, Pieter; Deforche, Koen; Edwards, Dustin; de Albuquerque-Junior, Antonio Eduardo; Vandamme, Anne-Mieke; Galvao-Castro, Bernardo; Alcantara, Luiz Carlos Junior

2012-01-01

It is estimated that 15 to 20 million people are infected with the human T-cell lymphotropic virus type 1 (HTLV-1). At present, there are more than 2,000 unique HTLV-1 isolate sequences published. A central database to aggregate sequence information from a range of epidemiological aspects including HTLV-1 infections, pathogenesis, origins, and evolutionary dynamics would be useful to scientists and physicians worldwide. Described here, we have developed a database that collects and annotates sequence data and can be accessed through a user-friendly search interface. The HTLV-1 Molecular Epidemiology Database website is available at http://htlv1db.bahia.fiocruz.br/. All data was obtained from publications available at GenBank or through contact with the authors. The database was developed using Apache Webserver 2.1.6 and SGBD MySQL. The webpage interfaces were developed in HTML and sever-side scripting written in PHP. The HTLV-1 Molecular Epidemiology Database is hosted on the Gonçalo Moniz/FIOCRUZ Research Center server. There are currently 2,457 registered sequences with 2,024 (82.37%) of those sequences representing unique isolates. Of these sequences, 803 (39.67%) contain information about clinical status (TSP/HAM, 17.19%; ATL, 7.41%; asymptomatic, 12.89%; other diseases, 2.17%; and no information, 60.32%). Further, 7.26% of sequences contain information on patient gender while 5.23% of sequences provide the age of the patient. The HTLV-1 Molecular Epidemiology Database retrieves and stores annotated HTLV-1 proviral sequences from clinical, epidemiological, and geographical studies. The collected sequences and related information are now accessible on a publically available and user-friendly website. This open-access database will support clinical research and vaccine development related to viral genotype.

Molecular Characterization of Transgenic Events Using Next Generation Sequencing Approach.

PubMed

Guttikonda, Satish K; Marri, Pradeep; Mammadov, Jafar; Ye, Liang; Soe, Khaing; Richey, Kimberly; Cruse, James; Zhuang, Meibao; Gao, Zhifang; Evans, Clive; Rounsley, Steve; Kumpatla, Siva P

2016-01-01

Demand for the commercial use of genetically modified (GM) crops has been increasing in light of the projected growth of world population to nine billion by 2050. A prerequisite of paramount importance for regulatory submissions is the rigorous safety assessment of GM crops. One of the components of safety assessment is molecular characterization at DNA level which helps to determine the copy number, integrity and stability of a transgene; characterize the integration site within a host genome; and confirm the absence of vector DNA. Historically, molecular characterization has been carried out using Southern blot analysis coupled with Sanger sequencing. While this is a robust approach to characterize the transgenic crops, it is both time- and resource-consuming. The emergence of next-generation sequencing (NGS) technologies has provided highly sensitive and cost- and labor-effective alternative for molecular characterization compared to traditional Southern blot analysis. Herein, we have demonstrated the successful application of both whole genome sequencing and target capture sequencing approaches for the characterization of single and stacked transgenic events and compared the results and inferences with traditional method with respect to key criteria required for regulatory submissions.

Ancient dna from pleistocene fossils: Preservation, recovery, and utility of ancient genetic information for quaternary research

NASA Astrophysics Data System (ADS)

Yang, Hong

Until recently, recovery and analysis of genetic information encoded in ancient DNA sequences from Pleistocene fossils were impossible. Recent advances in molecular biology offered technical tools to obtain ancient DNA sequences from well-preserved Quaternary fossils and opened the possibilities to directly study genetic changes in fossil species to address various biological and paleontological questions. Ancient DNA studies involving Pleistocene fossil material and ancient DNA degradation and preservation in Quaternary deposits are reviewed. The molecular technology applied to isolate, amplify, and sequence ancient DNA is also presented. Authentication of ancient DNA sequences and technical problems associated with modern and ancient DNA contamination are discussed. As illustrated in recent studies on ancient DNA from proboscideans, it is apparent that fossil DNA sequence data can shed light on many aspects of Quaternary research such as systematics and phylogeny. conservation biology, evolutionary theory, molecular taphonomy, and forensic sciences. Improvement of molecular techniques and a better understanding of DNA degradation during fossilization are likely to build on current strengths and to overcome existing problems, making fossil DNA data a unique source of information for Quaternary scientists.

Biological intuition in alignment-free methods: response to Posada.

PubMed

Ragan, Mark A; Chan, Cheong Xin

2013-08-01

A recent editorial in Journal of Molecular Evolution highlights opportunities and challenges facing molecular evolution in the era of next-generation sequencing. Abundant sequence data should allow more-complex models to be fit at higher confidence, making phylogenetic inference more reliable and improving our understanding of evolution at the molecular level. However, concern that approaches based on multiple sequence alignment may be computationally infeasible for large datasets is driving the development of so-called alignment-free methods for sequence comparison and phylogenetic inference. The recent editorial characterized these approaches as model-free, not based on the concept of homology, and lacking in biological intuition. We argue here that alignment-free methods have not abandoned models or homology, and can be biologically intuitive.

Reconstructing (super)trees from data sets with missing distances: not all is lost.

PubMed

Kettleborough, George; Dicks, Jo; Roberts, Ian N; Huber, Katharina T

2015-06-01

The wealth of phylogenetic information accumulated over many decades of biological research, coupled with recent technological advances in molecular sequence generation, presents significant opportunities for researchers to investigate relationships across and within the kingdoms of life. However, to make best use of this data wealth, several problems must first be overcome. One key problem is finding effective strategies to deal with missing data. Here, we introduce Lasso, a novel heuristic approach for reconstructing rooted phylogenetic trees from distance matrices with missing values, for data sets where a molecular clock may be assumed. Contrary to other phylogenetic methods on partial data sets, Lasso possesses desirable properties such as its reconstructed trees being both unique and edge-weighted. These properties are achieved by Lasso restricting its leaf set to a large subset of all possible taxa, which in many practical situations is the entire taxa set. Furthermore, the Lasso approach is distance-based, rendering it very fast to run and suitable for data sets of all sizes, including large data sets such as those generated by modern Next Generation Sequencing technologies. To better understand the performance of Lasso, we assessed it by means of artificial and real biological data sets, showing its effectiveness in the presence of missing data. Furthermore, by formulating the supermatrix problem as a particular case of the missing data problem, we assessed Lasso's ability to reconstruct supertrees. We demonstrate that, although not specifically designed for such a purpose, Lasso performs better than or comparably with five leading supertree algorithms on a challenging biological data set. Finally, we make freely available a software implementation of Lasso so that researchers may, for the first time, perform both rooted tree and supertree reconstruction with branch lengths on their own partial data sets. © The Author 2015. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.

Molecular characterization of a wild poliovirus type 3 epidemic in The Netherlands (1992 and 1993).

PubMed Central

Mulders, M N; van Loon, A M; van der Avoort, H G; Reimerink, J H; Ras, A; Bestebroer, T M; Drebot, M A; Kew, O M; Koopmans, M P

1995-01-01

An outbreak of poliomyelitis due to wild poliovirus type 3 (PV3) occurred in an unvaccinated community in The Netherlands between September 1992 and February 1993. The outbreak involved 71 patients. The aim of this study was to characterize the virus at the molecular level and to analyze the molecular evolution of the epidemic virus. Molecular analysis was carried out by sequencing the VP1/2A junction region (150 nucleotides) of 50 PV3 strains isolated in association with this outbreak and the entire VP1 gene of 14 strains. In addition, the sequence of the VP1/2A junction region of strains from geographical regions endemic for PV3 (Egypt, India, and Central Asia) was analyzed and compared with the nucleotide sequence of the epidemic strain from The Netherlands. The earliest isolate was obtained from river water sampled 3 weeks before diagnosis of the first poliomyelitis patient and was found by VP1/2A sequence analysis to be genetically identical to the strain isolated from the first patient. Sequence divergence among the strains from the epidemic in The Netherlands was less than 2%. The closest genetic similarity (97.3%) was found with an Indian isolate (New Delhi, December 1991), indicating the likely source of the virus. A more than 99% sequence similarity was found in the VP1/2A region. Finally, the sequence information was used to design primers for the specific and highly sensitive molecular detection of PV3 strains during the epidemic. PMID:8586711

A Predictive Model of Intein Insertion Site for Use in the Engineering of Molecular Switches

PubMed Central

Apgar, James; Ross, Mary; Zuo, Xiao; Dohle, Sarah; Sturtevant, Derek; Shen, Binzhang; de la Vega, Humberto; Lessard, Philip; Lazar, Gabor; Raab, R. Michael

2012-01-01

Inteins are intervening protein domains with self-splicing ability that can be used as molecular switches to control activity of their host protein. Successfully engineering an intein into a host protein requires identifying an insertion site that permits intein insertion and splicing while allowing for proper folding of the mature protein post-splicing. By analyzing sequence and structure based properties of native intein insertion sites we have identified four features that showed significant correlation with the location of the intein insertion sites, and therefore may be useful in predicting insertion sites in other proteins that provide native-like intein function. Three of these properties, the distance to the active site and dimer interface site, the SVM score of the splice site cassette, and the sequence conservation of the site showed statistically significant correlation and strong predictive power, with area under the curve (AUC) values of 0.79, 0.76, and 0.73 respectively, while the distance to secondary structure/loop junction showed significance but with less predictive power (AUC of 0.54). In a case study of 20 insertion sites in the XynB xylanase, two features of native insertion sites showed correlation with the splice sites and demonstrated predictive value in selecting non-native splice sites. Structural modeling of intein insertions at two sites highlighted the role that the insertion site location could play on the ability of the intein to modulate activity of the host protein. These findings can be used to enrich the selection of insertion sites capable of supporting intein splicing and hosting an intein switch. PMID:22649521

Gene expression distribution deconvolution in single-cell RNA sequencing.

PubMed

Wang, Jingshu; Huang, Mo; Torre, Eduardo; Dueck, Hannah; Shaffer, Sydney; Murray, John; Raj, Arjun; Li, Mingyao; Zhang, Nancy R

2018-06-26

Single-cell RNA sequencing (scRNA-seq) enables the quantification of each gene's expression distribution across cells, thus allowing the assessment of the dispersion, nonzero fraction, and other aspects of its distribution beyond the mean. These statistical characterizations of the gene expression distribution are critical for understanding expression variation and for selecting marker genes for population heterogeneity. However, scRNA-seq data are noisy, with each cell typically sequenced at low coverage, thus making it difficult to infer properties of the gene expression distribution from raw counts. Based on a reexamination of nine public datasets, we propose a simple technical noise model for scRNA-seq data with unique molecular identifiers (UMI). We develop deconvolution of single-cell expression distribution (DESCEND), a method that deconvolves the true cross-cell gene expression distribution from observed scRNA-seq counts, leading to improved estimates of properties of the distribution such as dispersion and nonzero fraction. DESCEND can adjust for cell-level covariates such as cell size, cell cycle, and batch effects. DESCEND's noise model and estimation accuracy are further evaluated through comparisons to RNA FISH data, through data splitting and simulations and through its effectiveness in removing known batch effects. We demonstrate how DESCEND can clarify and improve downstream analyses such as finding differentially expressed genes, identifying cell types, and selecting differentiation markers. Copyright © 2018 the Author(s). Published by PNAS.

An artificial molecular machine that builds an asymmetric catalyst

NASA Astrophysics Data System (ADS)

De Bo, Guillaume; Gall, Malcolm A. Y.; Kuschel, Sonja; De Winter, Julien; Gerbaux, Pascal; Leigh, David A.

2018-05-01

Biomolecular machines perform types of complex molecular-level tasks that artificial molecular machines can aspire to. The ribosome, for example, translates information from the polymer track it traverses (messenger RNA) to the new polymer it constructs (a polypeptide)1. The sequence and number of codons read determines the sequence and number of building blocks incorporated into the biomachine-synthesized polymer. However, neither control of sequence2,3 nor the transfer of length information from one polymer to another (which to date has only been accomplished in man-made systems through template synthesis)4 is easily achieved in the synthesis of artificial macromolecules. Rotaxane-based molecular machines5-7 have been developed that successively add amino acids8-10 (including β-amino acids10) to a growing peptide chain by the action of a macrocycle moving along a mono-dispersed oligomeric track derivatized with amino-acid phenol esters. The threaded macrocycle picks up groups that block its path and links them through successive native chemical ligation reactions11 to form a peptide sequence corresponding to the order of the building blocks on the track. Here, we show that as an alternative to translating sequence information, a rotaxane molecular machine can transfer the narrow polydispersity of a leucine-ester-derivatized polystyrene chain synthesized by atom transfer radical polymerization12 to a molecular-machine-made homo-leucine oligomer. The resulting narrow-molecular-weight oligomer folds to an α-helical secondary structure13 that acts as an asymmetric catalyst for the Juliá-Colonna epoxidation14,15 of chalcones.

Molecular cloning, expression and purification of L-amino acid oxidase from the Malayan pit viper Calloselasma rhodostoma.

PubMed

Kommoju, Phaneeswara Rao; Macheroux, Peter; Ghisla, Sandro

2007-03-01

A cDNA encoding LAAO from the Malayan pit viper (Calloselasma rhodostoma) was cloned into an expression vector of the methylotropic yeast Pichia pastoris. The LAAO open reading frame was inserted after the alpha-MF-signal sequence. Upon induction soluble and active LAAO is produced and exported into the culture supernatant at a concentration of up to 0.4 mg/L. Recombinant LAAO was purified from this by ion exchange and molecular sieve chromatography to yield apparently homogeneous protein in quantities of approximately 0.25 mg/L growth medium. Expressed LAAO exhibits the same electrophoretic mobility as native LAAO (62 kDa) and exhibits approximately the same extent of glycosylation as authentic LAAO from snake venom. Catalytic properties and substrate specificity of recombinant LAAO are similar to those of native enzyme.

Structural Prediction and In Silico Physicochemical Characterization for Mouse Caltrin I and Bovine Caltrin Proteins

PubMed Central

Grasso, Ernesto J.; Sottile, Adolfo E.; Coronel, Carlos E.

2016-01-01

It is known that caltrin (calcium transport inhibitor) protein binds to sperm cells during ejaculation and inhibits extracellular Ca2+ uptake. Although the sequence and some biological features of mouse caltrin I and bovine caltrin are known, their physicochemical properties and tertiary structure are mainly unknown. We predicted the 3D structures of mouse caltrin I and bovine caltrin by molecular homology modeling and threading. Surface electrostatic potentials and electric fields were calculated using the Poisson–Boltzmann equation. Several different bioinformatics tools and available web servers were used to thoroughly analyze the physicochemical characteristics of both proteins, such as their Kyte and Doolittle hydropathy scores and helical wheel projections. The results presented in this work significantly aid further understanding of the molecular mechanisms of caltrin proteins modulating physiological processes associated with fertilization. PMID:27812283

A Look Inside HIV Resistance through Retroviral Protease Interaction Maps

PubMed Central

Kontijevskis, Aleksejs; Prusis, Peteris; Petrovska, Ramona; Yahorava, Sviatlana; Mutulis, Felikss; Mutule, Ilze; Komorowski, Jan; Wikberg, Jarl E. S

2007-01-01

Retroviruses affect a large number of species, from fish and birds to mammals and humans, with global socioeconomic negative impacts. Here the authors report and experimentally validate a novel approach for the analysis of the molecular networks that are involved in the recognition of substrates by retroviral proteases. Using multivariate analysis of the sequence-based physiochemical descriptions of 61 retroviral proteases comprising wild-type proteases, natural mutants, and drug-resistant forms of proteases from nine different viral species in relation to their ability to cleave 299 substrates, the authors mapped the physicochemical properties and cross-dependencies of the amino acids of the proteases and their substrates, which revealed a complex molecular interaction network of substrate recognition and cleavage. The approach allowed a detailed analysis of the molecular–chemical mechanisms involved in substrate cleavage by retroviral proteases. PMID:17352531

Sequence-dependent nucleosome sliding in rotation-coupled and uncoupled modes revealed by molecular simulations

PubMed Central

Tan, Cheng; Takada, Shoji

2017-01-01

While nucleosome positioning on eukaryotic genome play important roles for genetic regulation, molecular mechanisms of nucleosome positioning and sliding along DNA are not well understood. Here we investigated thermally-activated spontaneous nucleosome sliding mechanisms developing and applying a coarse-grained molecular simulation method that incorporates both long-range electrostatic and short-range hydrogen-bond interactions between histone octamer and DNA. The simulations revealed two distinct sliding modes depending on the nucleosomal DNA sequence. A uniform DNA sequence showed frequent sliding with one base pair step in a rotation-coupled manner, akin to screw-like motions. On the contrary, a strong positioning sequence, the so-called 601 sequence, exhibits rare, abrupt transitions of five and ten base pair steps without rotation. Moreover, we evaluated the importance of hydrogen bond interactions on the sliding mode, finding that strong and weak bonds favor respectively the rotation-coupled and -uncoupled sliding movements. PMID:29194442

Structure and stability insights into tumour suppressor p53 evolutionary related proteins.

PubMed

Pagano, Bruno; Jama, Abdullah; Martinez, Pierre; Akanho, Ester; Bui, Tam T T; Drake, Alex F; Fraternali, Franca; Nikolova, Penka V

2013-01-01

The p53 family of genes and their protein products, namely, p53, p63 and p73, have over one billion years of evolutionary history. Advances in computational biology and genomics are enabling studies of the complexities of the molecular evolution of p53 protein family to decipher the underpinnings of key biological conditions spanning from cancer through to various metabolic and developmental disorders and facilitate the design of personalised medicines. However, a complete understanding of the inherent nature of the thermodynamic and structural stability of the p53 protein family is still lacking. This is due, to a degree, to the lack of comprehensive structural information for a large number of homologous proteins and to an incomplete knowledge of the intrinsic factors responsible for their stability and how these might influence function. Here we investigate the thermal stability, secondary structure and folding properties of the DNA-binding domains (DBDs) of a range of proteins from the p53 family using biophysical methods. While the N- and the C-terminal domains of the p53 family show sequence diversity and are normally targets for post-translational modifications and alternative splicing, the central DBD is highly conserved. Together with data obtained from Molecular Dynamics simulations in solution and with structure based homology modelling, our results provide further insights into the molecular properties of evolutionary related p53 proteins. We identify some marked structural differences within the p53 family, which could account for the divergence in biological functions as well as the subtleties manifested in the oligomerization properties of this family.

Protein Solubility and Protein Homeostasis: A Generic View of Protein Misfolding Disorders

PubMed Central

Vendruscolo, Michele; Knowles, Tuomas P.J.; Dobson, Christopher M.

2011-01-01

According to the “generic view” of protein aggregation, the ability to self-assemble into stable and highly organized structures such as amyloid fibrils is not an unusual feature exhibited by a small group of peptides and proteins with special sequence or structural properties, but rather a property shared by most proteins. At the same time, through a wide variety of techniques, many of which were originally devised for applications in other disciplines, it has also been established that the maintenance of proteins in a soluble state is a fundamental aspect of protein homeostasis. Taken together, these advances offer a unified framework for understanding the molecular basis of protein aggregation and for the rational development of therapeutic strategies based on the biological and chemical regulation of protein solubility. PMID:21825020

MultiSeq: unifying sequence and structure data for evolutionary analysis

PubMed Central

Roberts, Elijah; Eargle, John; Wright, Dan; Luthey-Schulten, Zaida

2006-01-01

Background Since the publication of the first draft of the human genome in 2000, bioinformatic data have been accumulating at an overwhelming pace. Currently, more than 3 million sequences and 35 thousand structures of proteins and nucleic acids are available in public databases. Finding correlations in and between these data to answer critical research questions is extremely challenging. This problem needs to be approached from several directions: information science to organize and search the data; information visualization to assist in recognizing correlations; mathematics to formulate statistical inferences; and biology to analyze chemical and physical properties in terms of sequence and structure changes. Results Here we present MultiSeq, a unified bioinformatics analysis environment that allows one to organize, display, align and analyze both sequence and structure data for proteins and nucleic acids. While special emphasis is placed on analyzing the data within the framework of evolutionary biology, the environment is also flexible enough to accommodate other usage patterns. The evolutionary approach is supported by the use of predefined metadata, adherence to standard ontological mappings, and the ability for the user to adjust these classifications using an electronic notebook. MultiSeq contains a new algorithm to generate complete evolutionary profiles that represent the topology of the molecular phylogenetic tree of a homologous group of distantly related proteins. The method, based on the multidimensional QR factorization of multiple sequence and structure alignments, removes redundancy from the alignments and orders the protein sequences by increasing linear dependence, resulting in the identification of a minimal basis set of sequences that spans the evolutionary space of the homologous group of proteins. Conclusion MultiSeq is a major extension of the Multiple Alignment tool that is provided as part of VMD, a structural visualization program for analyzing molecular dynamics simulations. Both are freely distributed by the NIH Resource for Macromolecular Modeling and Bioinformatics and MultiSeq is included with VMD starting with version 1.8.5. The MultiSeq website has details on how to download and use the software: PMID:16914055

Molecular evolution of ependymin and the phylogenetic resolution of early divergences among euteleost fishes.

PubMed

Ortí, G; Meyer, A

1996-04-01

The rate and pattern of DNA evolution of ependymin, a single-copy gene coding for a highly expressed glycoprotein in the brain matrix of teleost fishes, is characterized and its phylogenetic utility for fish systematics is assessed. DNA sequences were determined from catfish, electric fish, and characiforms and compared with published ependymin sequences from cyprinids, salmon, pike, and herring. Among these groups, ependymin amino acid sequences were highly divergent (up to 60% sequence difference), but had surprisingly similar hydropathy profiles and invariant glycosylation sites, suggesting that functional properties of the proteins are conserved. Comparison of base composition at third codon positions and introns revealed AT-rich introns and GC-rich third codon positions, suggesting that the biased codon usage observed might not be due to mutational bias. Phylogenetic information content of third codon positions was surprisingly high and sufficient to recover the most basal nodes of the tree, in spite of the observation that pairwise distances (at third codon positions) were well above the presumed saturation level. This finding can be explained by the high proportion of phylogenetically informative nonsynonymous changes at third codon positions among these highly divergent proteins. Ependymin DNA sequences have established the first molecular evidence for the monophyly of a group containing salmonids and esociforms. In addition, ependymin suggests a sister group relationship of electric fish (Gymnotiformes) and Characiformes, constituting a significant departure from currently accepted classifications. However, relationships among characiform lineages were not completely resolved by ependymin sequences in spite of seemingly appropriate levels of variation among taxa and considerably low levels of homoplasy in the data (consistency index = 0.7). If the diversification of Characiformes took place in an "explosive" manner, over a relatively short period of time this pattern should also be observed using other phylogenetic markers. Poor conservation of ependymin's primary structure hinders the design of efficient primers for PCR that could be used in wide-ranging fish systematic studies. However, alternative methods like PCR amplification from cDNA used here should provide promising comparative sequence data for the resolution of phylogenetic relationships among other basal lineages of teleost fishes.

Molecular Characterization of the S-Layer Gene, sbpA, of Bacillus sphaericus CCM 2177 and Production of a Functional S-Layer Fusion Protein with the Ability To Recrystallize in a Defined Orientation while Presenting the Fused Allergen

PubMed Central

Ilk, Nicola; Völlenkle, Christine; Egelseer, Eva M.; Breitwieser, Andreas; Sleytr, Uwe B.; Sára, Margit

2002-01-01

The nucleotide sequence encoding the crystalline bacterial cell surface (S-layer) protein SbpA of Bacillus sphaericus CCM 2177 was determined by a PCR-based technique using four overlapping fragments. The entire sbpA sequence indicated one open reading frame of 3,804 bp encoding a protein of 1,268 amino acids with a theoretical molecular mass of 132,062 Da and a calculated isoelectric point of 4.69. The N-terminal part of SbpA, which is involved in anchoring the S-layer subunits via a distinct type of secondary cell wall polymer to the rigid cell wall layer, comprises three S-layer-homologous motifs. For screening of amino acid positions located on the outer surface of the square S-layer lattice, the sequence encoding Strep-tag I, showing affinity to streptavidin, was linked to the 5′ end of the sequence encoding the recombinant S-layer protein (rSbpA) or a C-terminally truncated form (rSbpA31-1068). The deletion of 200 C-terminal amino acids did not interfere with the self-assembly properties of the S-layer protein but significantly increased the accessibility of Strep-tag I. Thus, the sequence encoding the major birch pollen allergen (Bet v1) was fused via a short linker to the sequence encoding the C-terminally truncated form rSpbA31-1068. Labeling of the square S-layer lattice formed by recrystallization of rSbpA31-1068/Bet v1 on peptidoglycan-containing sacculi with a Bet v1-specific monoclonal mouse antibody demonstrated the functionality of the fused protein sequence and its location on the outer surface of the S-layer lattice. The specific interactions between the N-terminal part of SbpA and the secondary cell wall polymer will be exploited for an oriented binding of the S-layer fusion protein on solid supports to generate regularly structured functional protein lattices. PMID:12089001

Long-term excretion of vaccine-derived poliovirus by a healthy child.

PubMed

Martín, Javier; Odoom, Kofi; Tuite, Gráinne; Dunn, Glynis; Hopewell, Nicola; Cooper, Gill; Fitzharris, Catherine; Butler, Karina; Hall, William W; Minor, Philip D

2004-12-01

A child was found to be excreting type 1 vaccine-derived poliovirus (VDPV) with a 1.1% sequence drift from Sabin type 1 vaccine strain in the VP1 coding region 6 months after he was immunized with oral live polio vaccine. Seventeen type 1 poliovirus isolates were recovered from stools taken from this child during the following 4 months. Contrary to expectation, the child was not deficient in humoral immunity and showed high levels of serum neutralization against poliovirus. Selected virus isolates were characterized in terms of their antigenic properties, virulence in transgenic mice, sensitivity for growth at high temperatures, and differences in nucleotide sequence from the Sabin type 1 strain. The VDPV isolates showed mutations at key nucleotide positions that correlated with the observed reversion to biological properties typical of wild polioviruses. A number of capsid mutations mapped at known antigenic sites leading to changes in the viral antigenic structure. Estimates of sequence evolution based on the accumulation of nucleotide changes in the VP1 coding region detected a "defective" molecular clock running at an apparent faster speed of 2.05% nucleotide changes per year versus 1% shown in previous studies. Remarkably, when compared to several type 1 VDPV strains of different origins, isolates from this child showed a much higher proportion of nonsynonymous versus synonymous nucleotide changes in the capsid coding region. This anomaly could explain the high VP1 sequence drift found and the ability of these virus strains to replicate in the gut for a longer period than expected.

Influence of Electron–Holes on DNA Sequence-Specific Mutation Rates

PubMed Central

Suárez-Villagrán, Martha Y; Azevedo, Ricardo B R; Miller, John H

2018-01-01

Abstract Biases in mutation rate can influence molecular evolution, yielding rates of evolution that vary widely in different parts of the genome and even among neighboring nucleotides. Here, we explore one possible mechanism of influence on sequence-specific mutation rates, the electron–hole, which can localize and potentially trigger a replication mismatch. A hole is a mobile site of positive charge created during one-electron oxidation by, for example, radiation, contact with a mutagenic agent, or oxidative stress. Its quantum wavelike properties cause it to localize at various sites with probabilities that vary widely, by orders of magnitude, and depend strongly on the local sequence. We find significant correlations between hole probabilities and mutation rates within base triplets, observed in published mutation accumulation experiments on four species of bacteria. We have also computed hole probability spectra for hypervariable segment I of the human mtDNA control region, which contains several mutational hotspots, and for heptanucleotides in noncoding regions of the human genome, whose polymorphism levels have recently been reported. We observe significant correlations between hole probabilities, and context-specific mutation and substitution rates. The correlation with hole probability cannot be explained entirely by CpG methylation in the heptanucleotide data. Peaks in hole probability tend to coincide with mutational hotspots, even in mtDNA where CpG methylation is rare. Our results suggest that hole-enhanced mutational mechanisms, such as oxidation-stabilized tautomerization and base deamination, contribute to molecular evolution. PMID:29617801

A Comprehensive, Automatically Updated Fungal ITS Sequence Dataset for Reference-Based Chimera Control in Environmental Sequencing Efforts.

PubMed

Nilsson, R Henrik; Tedersoo, Leho; Ryberg, Martin; Kristiansson, Erik; Hartmann, Martin; Unterseher, Martin; Porter, Teresita M; Bengtsson-Palme, Johan; Walker, Donald M; de Sousa, Filipe; Gamper, Hannes Andres; Larsson, Ellen; Larsson, Karl-Henrik; Kõljalg, Urmas; Edgar, Robert C; Abarenkov, Kessy

2015-01-01

The nuclear ribosomal internal transcribed spacer (ITS) region is the most commonly chosen genetic marker for the molecular identification of fungi in environmental sequencing and molecular ecology studies. Several analytical issues complicate such efforts, one of which is the formation of chimeric-artificially joined-DNA sequences during PCR amplification or sequence assembly. Several software tools are currently available for chimera detection, but rely to various degrees on the presence of a chimera-free reference dataset for optimal performance. However, no such dataset is available for use with the fungal ITS region. This study introduces a comprehensive, automatically updated reference dataset for fungal ITS sequences based on the UNITE database for the molecular identification of fungi. This dataset supports chimera detection throughout the fungal kingdom and for full-length ITS sequences as well as partial (ITS1 or ITS2 only) datasets. The performance of the dataset on a large set of artificial chimeras was above 99.5%, and we subsequently used the dataset to remove nearly 1,000 compromised fungal ITS sequences from public circulation. The dataset is available at http://unite.ut.ee/repository.php and is subject to web-based third-party curation.

String Mining in Bioinformatics

NASA Astrophysics Data System (ADS)

Abouelhoda, Mohamed; Ghanem, Moustafa

Sequence analysis is a major area in bioinformatics encompassing the methods and techniques for studying the biological sequences, DNA, RNA, and proteins, on the linear structure level. The focus of this area is generally on the identification of intra- and inter-molecular similarities. Identifying intra-molecular similarities boils down to detecting repeated segments within a given sequence, while identifying inter-molecular similarities amounts to spotting common segments among two or multiple sequences. From a data mining point of view, sequence analysis is nothing but string- or pattern mining specific to biological strings. For a long time, this point of view, however, has not been explicitly embraced neither in the data mining nor in the sequence analysis text books, which may be attributed to the co-evolution of the two apparently independent fields. In other words, although the word "data-mining" is almost missing in the sequence analysis literature, its basic concepts have been implicitly applied. Interestingly, recent research in biological sequence analysis introduced efficient solutions to many problems in data mining, such as querying and analyzing time series [49,53], extracting information from web pages [20], fighting spam mails [50], detecting plagiarism [22], and spotting duplications in software systems [14].

String Mining in Bioinformatics

NASA Astrophysics Data System (ADS)

Abouelhoda, Mohamed; Ghanem, Moustafa

Sequence analysis is a major area in bioinformatics encompassing the methods and techniques for studying the biological sequences, DNA, RNA, and proteins, on the linear structure level. The focus of this area is generally on the identification of intra- and inter-molecular similarities. Identifying intra-molecular similarities boils down to detecting repeated segments within a given sequence, while identifying inter-molecular similarities amounts to spotting common segments among two or multiple sequences. From a data mining point of view, sequence analysis is nothing but string- or pattern mining specific to biological strings. For a long time, this point of view, however, has not been explicitly embraced neither in the data mining nor in the sequence analysis text books, which may be attributed to the co-evolution of the two apparently independent fields. In other words, although the word “data-mining” is almost missing in the sequence analysis literature, its basic concepts have been implicitly applied. Interestingly, recent research in biological sequence analysis introduced efficient solutions to many problems in data mining, such as querying and analyzing time series [49,53], extracting information from web pages [20], fighting spam mails [50], detecting plagiarism [22], and spotting duplications in software systems [14].

Influence of Molecular Resolution on Sequence-Based Discovery of Ecological Diversity among Synechococcus Populations in an Alkaline Siliceous Hot Spring Microbial Mat ▿ †

PubMed Central

Melendrez, Melanie C.; Lange, Rachel K.; Cohan, Frederick M.; Ward, David M.

2011-01-01

Previous research has shown that sequences of 16S rRNA genes and 16S-23S rRNA internal transcribed spacer regions may not have enough genetic resolution to define all ecologically distinct Synechococcus populations (ecotypes) inhabiting alkaline, siliceous hot spring microbial mats. To achieve higher molecular resolution, we studied sequence variation in three protein-encoding loci sampled by PCR from 60°C and 65°C sites in the Mushroom Spring mat (Yellowstone National Park, WY). Sequences were analyzed using the ecotype simulation (ES) and AdaptML algorithms to identify putative ecotypes. Between 4 and 14 times more putative ecotypes were predicted from variation in protein-encoding locus sequences than from variation in 16S rRNA and 16S-23S rRNA internal transcribed spacer sequences. The number of putative ecotypes predicted depended on the number of sequences sampled and the molecular resolution of the locus. Chao estimates of diversity indicated that few rare ecotypes were missed. Many ecotypes hypothesized by sequence analyses were different in their habitat specificities, suggesting different adaptations to temperature or other parameters that vary along the flow channel. PMID:21169433

A Comprehensive, Automatically Updated Fungal ITS Sequence Dataset for Reference-Based Chimera Control in Environmental Sequencing Efforts

PubMed Central

Nilsson, R. Henrik; Tedersoo, Leho; Ryberg, Martin; Kristiansson, Erik; Hartmann, Martin; Unterseher, Martin; Porter, Teresita M.; Bengtsson-Palme, Johan; Walker, Donald M.; de Sousa, Filipe; Gamper, Hannes Andres; Larsson, Ellen; Larsson, Karl-Henrik; Kõljalg, Urmas; Edgar, Robert C.; Abarenkov, Kessy

2015-01-01

The nuclear ribosomal internal transcribed spacer (ITS) region is the most commonly chosen genetic marker for the molecular identification of fungi in environmental sequencing and molecular ecology studies. Several analytical issues complicate such efforts, one of which is the formation of chimeric—artificially joined—DNA sequences during PCR amplification or sequence assembly. Several software tools are currently available for chimera detection, but rely to various degrees on the presence of a chimera-free reference dataset for optimal performance. However, no such dataset is available for use with the fungal ITS region. This study introduces a comprehensive, automatically updated reference dataset for fungal ITS sequences based on the UNITE database for the molecular identification of fungi. This dataset supports chimera detection throughout the fungal kingdom and for full-length ITS sequences as well as partial (ITS1 or ITS2 only) datasets. The performance of the dataset on a large set of artificial chimeras was above 99.5%, and we subsequently used the dataset to remove nearly 1,000 compromised fungal ITS sequences from public circulation. The dataset is available at http://unite.ut.ee/repository.php and is subject to web-based third-party curation. PMID:25786896

Cloning, sequencing, and expression of the gene coding for bile acid 7 alpha-hydroxysteroid dehydrogenase from Eubacterium sp. strain VPI 12708.

PubMed Central

Baron, S F; Franklund, C V; Hylemon, P B

1991-01-01

Southern blot analysis indicated that the gene encoding the constitutive, NADP-linked bile acid 7 alpha-hydroxysteroid dehydrogenase of Eubacterium sp. strain VPI 12708 was located on a 6.5-kb EcoRI fragment of the chromosomal DNA. This fragment was cloned into bacteriophage lambda gt11, and a 2.9-kb piece of this insert was subcloned into pUC19, yielding the recombinant plasmid pBH51. DNA sequence analysis of the 7 alpha-hydroxysteroid dehydrogenase gene in pBH51 revealed a 798-bp open reading frame, coding for a protein with a calculated molecular weight of 28,500. A putative promoter sequence and ribosome binding site were identified. The 7 alpha-hydroxysteroid dehydrogenase mRNA transcript in Eubacterium sp. strain VPI 12708 was about 0.94 kb in length, suggesting that it is monocistronic. An Escherichia coli DH5 alpha transformant harboring pBH51 had approximately 30-fold greater levels of 7 alpha-hydroxysteroid dehydrogenase mRNA, immunoreactive protein, and specific activity than Eubacterium sp. strain VPI 12708. The 7 alpha-hydroxysteroid dehydrogenase purified from the pBH51 transformant was similar in subunit molecular weight, specific activity, and kinetic properties to that from Eubacterium sp. strain VPI 12708, and it reached with antiserum raised against the authentic enzyme on Western immunoblots. Alignment of the amino acid sequence of the 7 alpha-hydroxysteroid dehydrogenase with those of 10 other pyridine nucleotide-linked alcohol/polyol dehydrogenases revealed six conserved amino acid residues in the N-terminal regions thought to function in coenzyme binding. Images PMID:1856160

The long tail of molecular alterations in non-small cell lung cancer: a single-institution experience of next-generation sequencing in clinical molecular diagnostics.

PubMed

Fumagalli, Caterina; Vacirca, Davide; Rappa, Alessandra; Passaro, Antonio; Guarize, Juliana; Rafaniello Raviele, Paola; de Marinis, Filippo; Spaggiari, Lorenzo; Casadio, Chiara; Viale, Giuseppe; Barberis, Massimo; Guerini-Rocco, Elena

2018-03-13

Molecular profiling of advanced non-small cell lung cancers (NSCLC) is essential to identify patients who may benefit from targeted treatments. In the last years, the number of potentially actionable molecular alterations has rapidly increased. Next-generation sequencing allows for the analysis of multiple genes simultaneously. To evaluate the feasibility and the throughput of next-generation sequencing in clinical molecular diagnostics of advanced NSCLC. A single-institution cohort of 535 non-squamous NSCLC was profiled using a next-generation sequencing panel targeting 22 actionable and cancer-related genes. 441 non-squamous NSCLC (82.4%) harboured at least one gene alteration, including 340 cases (63.6%) with clinically relevant molecular aberrations. Mutations have been detected in all but one gene ( FGFR1 ) of the panel. Recurrent alterations were observed in KRAS , TP53 , EGFR , STK11 and MET genes, whereas the remaining genes were mutated in <5% of the cases. Concurrent mutations were detected in 183 tumours (34.2%), mostly impairing KRAS or EGFR in association with TP53 alterations. The study highlights the feasibility of targeted next-generation sequencing in clinical setting. The majority of NSCLC harboured mutations in clinically relevant genes, thus identifying patients who might benefit from different targeted therapies. © Article author(s) (or their employer(s) unless otherwise stated in the text of the article) 2018. All rights reserved. No commercial use is permitted unless otherwise expressly granted.

Raman spectroscopy as a tool to investigate the structure and electronic properties of carbon-atom wires

PubMed Central

Milani, Alberto; Tommasini, Matteo; Russo, Valeria; Li Bassi, Andrea; Lucotti, Andrea; Cataldo, Franco

2015-01-01

Summary Graphene, nanotubes and other carbon nanostructures have shown potential as candidates for advanced technological applications due to the different coordination of carbon atoms and to the possibility of π-conjugation. In this context, atomic-scale wires comprised of sp-hybridized carbon atoms represent ideal 1D systems to potentially downscale devices to the atomic level. Carbon-atom wires (CAWs) can be arranged in two possible structures: a sequence of double bonds (cumulenes), resulting in a 1D metal, or an alternating sequence of single–triple bonds (polyynes), expected to show semiconducting properties. The electronic and optical properties of CAWs can be finely tuned by controlling the wire length (i.e., the number of carbon atoms) and the type of termination (e.g., atom, molecular group or nanostructure). Although linear, sp-hybridized carbon systems are still considered elusive and unstable materials, a number of nanostructures consisting of sp-carbon wires have been produced and characterized to date. In this short review, we present the main CAW synthesis techniques and stabilization strategies and we discuss the current status of the understanding of their structural, electronic and vibrational properties with particular attention to how these properties are related to one another. We focus on the use of vibrational spectroscopy to provide information on the structural and electronic properties of the system (e.g., determination of wire length). Moreover, by employing Raman spectroscopy and surface enhanced Raman scattering in combination with the support of first principles calculations, we show that a detailed understanding of the charge transfer between CAWs and metal nanoparticles may open the possibility to tune the electronic structure from alternating to equalized bonds. PMID:25821689

From Peptides to Proteins: Systematic Control of Net Molecular Charge and Hydrophilicity on the Kinetics of Calcite Growth

NASA Astrophysics Data System (ADS)

Elhadj, S.; de Yoreo, J. J.; Hoyer, J. J.; Dove, P. M.

2006-12-01

The compartment-specific compositions of biologic molecules isolated from biominerals suggest that control of mineral growth may be linked to biochemical features. Here we define a systematic relationship between the ability of biomolecules in solution to promote the growth of calcite (CaCO3) and their net negative molecular charge and hydrophilicity. The degree of enhancement is dependent on peptide composition, but not on peptide sequence. Data analysis shows that this rate enhancement arises from an increase in the kinetic coefficient. We interpret the mechanism of growth enhancement to be a catalytic process whereby biomolecules reduce the magnitude of the diffusive barrier, Ek, by perturbations that displace water molecules- a water shell destruction mechanism. The result is a decrease in the repulsive barrier for attachment of solutes to the solid phase. This previously unrecognized relationship also rationalizes recently reported data showing acceleration of calcite growth rates over rates measured in the pure system by nanomolar levels of abalone nacre proteins. These findings show that the growth-modifying properties of small model peptides may be scaled up to analyze mineralization processes that are mediated by more complex proteins. We suggest that enhancement of calcite growth may now be estimated a priori from the composition of peptide sequences and the calculated values of hydrophilicity and net molecular charge without need for detailed tests for each biomolecule. This insight may contribute to an improved understanding of mineralization in diverse systems of biomineralization.

Receptors and aging: structural selectivity of the rhamnose-receptor on fibroblasts as shown by Ca(2+)-mobilization and gene-expression profiles.

PubMed

Faury, G; Molinari, J; Rusova, E; Mariko, B; Raveaud, S; Huber, P; Velebny, V; Robert, A M; Robert, L

2011-01-01

Qualitative and quantitative modifications of receptors were shown to play a key role in cell and tissue aging. We recently described the properties of a rhamnose-recognizing receptor on fibroblasts involved in the mediation of age-dependent functions of these cells. Using Ca(2+)-mobilization and DNA-microarrays we could show in the presence of rhamnose-rich oligo- and polysaccharides (RROPs) Ca(2+)-mobilization and changes in gene regulation. Here, we compared the effects of several RROPs, differing in their carbohydrate sequence and molecular weights, in normal human dermal fibroblasts (NHDFs). It appeared that different structural features were required for maximal effects on Ca(2+)-mobilization and gene-expression profiles. Maximal effect on Ca(2+) influx and intracellular free calcium regulation was exhibited by RROP-1, a 50 kDa average molecular weight polysaccharide, and RROP-3, a 5 kDa average molecular weight oligosaccharide with a different carbohydrate sequence. Maximal effect on gene-expression profiles was obtained with RROP-3. These results suggest the possibility of several different transmission pathways from the rhamnose-receptor to intracellular targets, differentially affecting these two intracellular functions, with potential consequences on aging. Although of only relative specificity, this receptor site exhibits a high affinity for rhamnose, absent from vertebrate glycoconjugates. The rhamnose-receptor might well represent an evolutionary conserved conformation of a prokaryote lectin. Copyright © 2010 Elsevier Ireland Ltd. All rights reserved.

A molecular epidemiology study based on VP2 gene sequences reveals that a new genotype of infectious bursal disease virus is dominantly prevalent in Italy.

PubMed

Lupini, Caterina; Giovanardi, Davide; Pesente, Patrizia; Bonci, Michela; Felice, Viviana; Rossi, Giulia; Morandini, Emilio; Cecchinato, Mattia; Catelli, Elena

2016-08-01

A distinctive infectious bursal disease (IBD) virus genotype (ITA) was detected in IBD-live vaccinated broilers in Italy without clinical signs of IBD. It was isolated in specific-pathogen-free eggs and molecularly characterized in the hypervariable region of the virus protein (VP) 2. Phylogenetic analysis showed that ITA strains clustered separately from other homologous reference sequences of IBDVs, either classical or very virulent, retrieved from GenBank or previously reported in Italy, and from vaccine strains. The new genotype shows peculiar molecular characteristics in key positions of the VP2 hypervariable region, which affect charged or potentially glycosylated amino acids virtually associated with important changes in virus properties. Characterization of 41 IBDV strains detected in Italy between 2013 and 2014 showed that ITA is emergent in densely populated poultry areas of Italy, being 68% of the IBDV detections made during routine diagnostic activity over a two-year period, in spite of the immunity induced by large-scale vaccination. Four very virulent strains (DV86) and one classical strain (HPR2), together with eight vaccine strains, were also detected. The currently available epidemiological and clinical data do not allow the degree of pathogenicity of the ITA genotype to be defined. Only in vivo experimental pathogenicity studies conducted in secure isolation conditions, through the evaluation of clinical signs and macro/microscopic lesions, will clarify conclusively the virulence of the new Italian genotype.

Purification of a factor from the granules of a rat natural killer cell line (RNK) that reduces tumor cell growth and changes tumor morphology. Molecular identity with a granule serine protease (RNKP-1).

PubMed

Sayers, T J; Wiltrout, T A; Sowder, R; Munger, W L; Smyth, M J; Henderson, L E

1992-01-01

We have purified a protein from the granules of the rat NK leukemia cell line (RNK) that is cytostatic to a variety of tumor cells. This protein shows no species specificity because certain tumor cell lines of mouse, rat, and human origin were equally sensitive to its growth inhibitory effects. Treatment of sensitive cells resulted in a rounding of the cells followed by homotypic aggregation into large aggregates. The granule protein was distinct from cytolysin, Na-Cbz-Lys-thiobenzylester-esterase, or leukolexin. It had a molecular mass of 29 to 31 kDa, bound strongly to heparin, was inactivated by heating at 70 degrees C for 5 min or reduction, but was stable to trypsin treatment. By using molecular sieve chromatography, heparin agarose chromatography, and reverse phase HPLC, this protein was purified to homogeneity. The first 33 amino acids of the N-terminal amino acid sequence showed complete identity to the sequence predicted from a rat serine protease gene recently cloned and designated RNKP-1. Therefore we have purified a novel serine protease and demonstrated that it has effects on the growth and morphology of certain tumor cells. Other serine proteases that were structurally related and have substantial homology with RNKP-1 at the amino acid level showed neither growth inhibitory properties nor affected the morphology of the tumor target cells we used.

Molecular Structure and Sequence in Complex Coacervates

NASA Astrophysics Data System (ADS)

Sing, Charles; Lytle, Tyler; Madinya, Jason; Radhakrishna, Mithun

Oppositely-charged polyelectrolytes in aqueous solution can undergo associative phase separation, in a process known as complex coacervation. This results in a polyelectrolyte-dense phase (coacervate) and polyelectrolyte-dilute phase (supernatant). There remain challenges in understanding this process, despite a long history in polymer physics. We use Monte Carlo simulation to demonstrate that molecular features (charge spacing, size) play a crucial role in governing the equilibrium in coacervates. We show how these molecular features give rise to strong monomer sequence effects, due to a combination of counterion condensation and correlation effects. We distinguish between structural and sequence-based correlations, which can be designed to tune the phase diagram of coacervation. Sequence effects further inform the physical understanding of coacervation, and provide the basis for new coacervation models that take monomer-level features into account.

Sequencing cDNAs: An Introduction to DNA Sequence Analysis in the Undergraduate Molecular Genetics Course.

ERIC Educational Resources Information Center

Galewsky, Samuel

2000-01-01

Introduces a series of molecular genetics laboratories where students pick a single colony from a Drosophila melanogester embryo cDNA library and purify the plasmid, then analyze the insert through restriction digests and gel electrophoresis. (Author/YDS)

New single-copy nuclear genes for scale insect systematics

USDA-ARS?s Scientific Manuscript database

Despite the advent of next-generation sequencing, the polymerase chain reaction (PCR) and Sanger sequencing remain useful tools for molecular identification and systematics. To date, molecular systematics of scale insects has been constrained by the paucity of loci that researchers have been able to...

Genomic sequencing and the impact of molecular diagnosis on patient care.

PubMed

Solomon, Benjamin D

2015-02-01

Evolving sequencing technologies allow more accurate, efficient and affordable genomic analysis. As a result, these technologies are increasingly available, especially to provide molecular diagnoses for patients with suspected genetic disorders. However, there are many challenges to using genomic sequencing to benefit patients, including concerns that there is insufficient evidence that identifying an underlying molecular explanation may positively impact a patient's healthcare. This concern has many repercussions, including funding and/or (in some countries and healthcare systems) insurance reimbursement for genomic sequencing. To investigate this concern, all monogenic disorders were analyzed based on the impact of achieving molecular diagnosis. Of the 2,849 individual genes in which germline mutations cause disorders (not including contiguous gene syndromes or what may be categorized as susceptibility alleles), our analyses showed a specific, available intervention related to at least one affected organ system for 1,419 (49.8%) genes. In 95.6% of these genes, the intervention(s) would be recommended during the pediatric time frame.

Cracking the Code of Human Diseases Using Next-Generation Sequencing: Applications, Challenges, and Perspectives

PubMed Central

Precone, Vincenza; Del Monaco, Valentina; Esposito, Maria Valeria; De Palma, Fatima Domenica Elisa; Ruocco, Anna; D'Argenio, Valeria

2015-01-01

Next-generation sequencing (NGS) technologies have greatly impacted on every field of molecular research mainly because they reduce costs and increase throughput of DNA sequencing. These features, together with the technology's flexibility, have opened the way to a variety of applications including the study of the molecular basis of human diseases. Several analytical approaches have been developed to selectively enrich regions of interest from the whole genome in order to identify germinal and/or somatic sequence variants and to study DNA methylation. These approaches are now widely used in research, and they are already being used in routine molecular diagnostics. However, some issues are still controversial, namely, standardization of methods, data analysis and storage, and ethical aspects. Besides providing an overview of the NGS-based approaches most frequently used to study the molecular basis of human diseases at DNA level, we discuss the principal challenges and applications of NGS in the field of human genomics. PMID:26665001

Role of Angiogenesis in the Etiology and Prevention of Ovarian Cancer

DTIC Science & Technology

2004-10-01

sequences of peptides were confirmed by N-terminal sequencing and viewer ( Molecular Simulations , Inc.) and were analyzed using X-PLOR mass spectrometry...clinical benefit seen in trials using Avastin (anti-VEGF humanized antibody) and chemotherapy. We are currently pursuing the molecular mechanism involved in...and ovarian cancer. Invited speaker. ASCO Molecular Therapeutics symposium. Nov. 8-10, 2002, San Diego, CA. 13. I. V. Subramanian, R. Ghebre, Y

Toolbox Approaches Using Molecular Markers and 16S rRNA Gene Amplicon Data Sets for Identification of Fecal Pollution in Surface Water

PubMed Central

Staley, C.; Sadowsky, M. J.; Gyawali, P.; Sidhu, J. P. S.; Palmer, A.; Beale, D. J.; Toze, S.

2015-01-01

In this study, host-associated molecular markers and bacterial 16S rRNA gene community analysis using high-throughput sequencing were used to identify the sources of fecal pollution in environmental waters in Brisbane, Australia. A total of 92 fecal and composite wastewater samples were collected from different host groups (cat, cattle, dog, horse, human, and kangaroo), and 18 water samples were collected from six sites (BR1 to BR6) along the Brisbane River in Queensland, Australia. Bacterial communities in the fecal, wastewater, and river water samples were sequenced. Water samples were also tested for the presence of bird-associated (GFD), cattle-associated (CowM3), horse-associated, and human-associated (HF183) molecular markers, to provide multiple lines of evidence regarding the possible presence of fecal pollution associated with specific hosts. Among the 18 water samples tested, 83%, 33%, 17%, and 17% were real-time PCR positive for the GFD, HF183, CowM3, and horse markers, respectively. Among the potential sources of fecal pollution in water samples from the river, DNA sequencing tended to show relatively small contributions from wastewater treatment plants (up to 13% of sequence reads). Contributions from other animal sources were rarely detected and were very small (<3% of sequence reads). Source contributions determined via sequence analysis versus detection of molecular markers showed variable agreement. A lack of relationships among fecal indicator bacteria, host-associated molecular markers, and 16S rRNA gene community analysis data was also observed. Nonetheless, we show that bacterial community and host-associated molecular marker analyses can be combined to identify potential sources of fecal pollution in an urban river. This study is a proof of concept, and based on the results, we recommend using bacterial community analysis (where possible) along with PCR detection or quantification of host-associated molecular markers to provide information on the sources of fecal pollution in waterways. PMID:26231650

Cyanine-based probe\\tag-peptide pair fluorescence protein imaging and fluorescence protein imaging methods

DOEpatents

Mayer-Cumblidge, M. Uljana; Cao, Haishi

2013-01-15

A molecular probe comprises two arsenic atoms and at least one cyanine based moiety. A method of producing a molecular probe includes providing a molecule having a first formula, treating the molecule with HgOAc, and subsequently transmetallizing with AsCl.sub.3. The As is liganded to ethanedithiol to produce a probe having a second formula. A method of labeling a peptide includes providing a peptide comprising a tag sequence and contacting the peptide with a biarsenical molecular probe. A complex is formed comprising the tag sequence and the molecular probe. A method of studying a peptide includes providing a mixture containing a peptide comprising a peptide tag sequence, adding a biarsenical probe to the mixture, and monitoring the fluorescence of the mixture.

Cyanine-based probe\\tag-peptide pair for fluorescence protein imaging and fluorescence protein imaging methods

DOEpatents

Mayer-Cumblidge, M Uljana [Richland, WA; Cao, Haishi [Richland, WA

2010-08-17

A molecular probe comprises two arsenic atoms and at least one cyanine based moiety. A method of producing a molecular probe includes providing a molecule having a first formula, treating the molecule with HgOAc, and subsequently transmetallizing with AsCl.sub.3. The As is liganded to ethanedithiol to produce a probe having a second formula. A method of labeling a peptide includes providing a peptide comprising a tag sequence and contacting the peptide with a biarsenical molecular probe. A complex is formed comprising the tag sequence and the molecular probe. A method of studying a peptide includes providing a mixture containing a peptide comprising a peptide tag sequence, adding a biarsenical probe to the mixture, and monitoring the fluorescence of the mixture.

Evolutional dynamics of 45S and 5S ribosomal DNA in ancient allohexaploid Atropa belladonna.

PubMed

Volkov, Roman A; Panchuk, Irina I; Borisjuk, Nikolai V; Hosiawa-Baranska, Marta; Maluszynska, Jolanta; Hemleben, Vera

2017-01-23

Polyploid hybrids represent a rich natural resource to study molecular evolution of plant genes and genomes. Here, we applied a combination of karyological and molecular methods to investigate chromosomal structure, molecular organization and evolution of ribosomal DNA (rDNA) in nightshade, Atropa belladonna (fam. Solanaceae), one of the oldest known allohexaploids among flowering plants. Because of their abundance and specific molecular organization (evolutionarily conserved coding regions linked to variable intergenic spacers, IGS), 45S and 5S rDNA are widely used in plant taxonomic and evolutionary studies. Molecular cloning and nucleotide sequencing of A. belladonna 45S rDNA repeats revealed a general structure characteristic of other Solanaceae species, and a very high sequence similarity of two length variants, with the only difference in number of short IGS subrepeats. These results combined with the detection of three pairs of 45S rDNA loci on separate chromosomes, presumably inherited from both tetraploid and diploid ancestor species, example intensive sequence homogenization that led to substitution/elimination of rDNA repeats of one parent. Chromosome silver-staining revealed that only four out of six 45S rDNA sites are frequently transcriptionally active, demonstrating nucleolar dominance. For 5S rDNA, three size variants of repeats were detected, with the major class represented by repeats containing all functional IGS elements required for transcription, the intermediate size repeats containing partially deleted IGS sequences, and the short 5S repeats containing severe defects both in the IGS and coding sequences. While shorter variants demonstrate increased rate of based substitution, probably in their transition into pseudogenes, the functional 5S rDNA variants are nearly identical at the sequence level, pointing to their origin from a single parental species. Localization of the 5S rDNA genes on two chromosome pairs further supports uniparental inheritance from the tetraploid progenitor. The obtained molecular, cytogenetic and phylogenetic data demonstrate complex evolutionary dynamics of rDNA loci in allohexaploid species of Atropa belladonna. The high level of sequence unification revealed in 45S and 5S rDNA loci of this ancient hybrid species have been seemingly achieved by different molecular mechanisms.

Factors affecting nucleolytic efficiency of some ternary metal complexes with DNA binding and recognition domains. Crystal and molecular structure of Zn(phen)(edda).

PubMed

Seng, Hoi-Ling; Ong, Han-Kiat Alan; Rahman, Raja Noor Zaliha Raja Abd; Yamin, Bohari M; Tiekink, Edward R T; Tan, Kong Wai; Maah, Mohd Jamil; Caracelli, Ignez; Ng, Chew Hee

2008-11-01

The binding selectivity of the M(phen)(edda) (M=Cu, Co, Ni, Zn; phen=1,10-phenanthroline, edda=ethylenediaminediacetic acid) complexes towards ds(CG)(6), ds(AT)(6) and ds(CGCGAATTCGCG) B-form oligonucleotide duplexes were studied by CD spectroscopy and molecular modeling. The binding mode is intercalation and there is selectivity towards AT-sequence and stacking preference for A/A parallel or diagonal adjacent base steps in their intercalation. The nucleolytic properties of these complexes were investigated and the factors affecting the extent of cleavage were determined to be: concentration of complex, the nature of metal(II) ion, type of buffer, pH of buffer, incubation time, incubation temperature, and the presence of hydrogen peroxide or ascorbic acid as exogenous reagents. The fluorescence property of these complexes and its origin were also investigated. The crystal structure of the Zn(phen)(edda) complex is reported in which the zinc atom displays a distorted trans-N(4)O(2) octahedral geometry; the crystal packing features double layers of complex molecules held together by extensive hydrogen bonding that inter-digitate with adjacent double layers via pi...pi interactions between 1,10-phenanthroline residues. The structure is compared with that of the recently described copper(II) analogue and, with the latter, included in molecular modeling.

An unconventional antimicrobial protein histone from freshwater prawn Macrobrachium rosenbergii: analysis of immune properties.

PubMed

Arockiaraj, Jesu; Gnanam, Annie J; Kumaresan, Venkatesh; Palanisamy, Rajesh; Bhatt, Prasanth; Thirumalai, Muthukumaresan Kuppusamy; Roy, Arpita; Pasupuleti, Mukesh; Kasi, Marimuthu

2013-11-01

In this study, we have reported the first histone characterized at molecular level from freshwater prawn Macrobrachium rosenbergii (MrHis). A full length cDNA of MrHis (751 base pairs) was identified from an established M. rosenbergii cDNA library using GS-FLX technique. It encodes 137 amino acid residues with a calculated molecular mass of 15 kDa and an isoelectric point of 10.5. MrHis peptide contains a histone H2A signature between 21 and 27 amino acids. Homologous analysis showed that MrHis had a significant sequence identity (99%) with other known histone H2A groups especially from Penaeus monodon. Phylogenetic analysis of MrHis showed a strong relationship with other amino acid sequences from histone H2A arthropod groups. Further phylogenetic analysis showed that the MrHis belongs to histone H2A superfamily and H2A1A sub-family. Secondary structure of MrHis showed that the protein contains 50.36% α-helical region and 49.64% coils. The 3D model of MrHis was predicted by I-Tasser program and the model was evaluated for quality analysis including C-score analysis, Ramachandran plot analysis and RMSD analysis. The surface view analysis of MrHis showed the active domain at the N terminal. The antimicrobial property of MrHis protein was confirmed by the helical structure and the total hydrophobic surface along with its net charge. The MFE of the predicted RNA structure of MrHis is -128.62 kcal/mol, shows its mRNA stability. Schiffer-Edmundson helical wheel analysis of the N-terminal of MrHis showed a perfect amphipathic nature of the peptide. Significantly (P < 0.05) highest gene expression was noticed in the hemocyte and is induced with viral (WSBV and MrNV) and bacteria (A eromonas hydrophila and Vibrio harveyi) infections. The coding sequence of recombinant MrHis protein was expressed in a pMAL vector and purified to study the antimicrobial properties. The recombinant product showed antimicrobial activity against both Gram negative and Gram positive bacteria. In this study, the recombinant MrHis protein displayed antimicrobial activity in its entirety. Hence, it is possible to suggest that the activity may be due to the direct defense role of histone or its N-terminal antimicrobial property. However, this remains to be verified by detailed investigations. Copyright © 2013 Elsevier Ltd. All rights reserved.

A RESTful application programming interface for the PubMLST molecular typing and genome databases

PubMed Central

Bray, James E.; Maiden, Martin C. J.

2017-01-01

Abstract Molecular typing is used to differentiate microorganisms at the subspecies or strain level for epidemiological investigations, infection control, public health and environmental sampling. DNA sequence-based typing methods require authoritative databases that link sequence variants to nomenclature in order to facilitate communication and comparison of identified types in national or global settings. The PubMLST website (https://pubmlst.org/) fulfils this role for over a hundred microorganisms for which it hosts curated molecular sequence typing data, providing sequence and allelic profile definitions for multi-locus sequence typing (MLST) and single-gene typing approaches. In recent years, these have expanded to cover the whole genome with schemes such as core genome MLST (cgMLST) and whole genome MLST (wgMLST) which catalogue the allelic diversity found in hundreds to thousands of genes. These approaches provide a common nomenclature for high-resolution strain characterization and comparison. Molecular typing information is linked to isolate provenance, phenotype, and increasingly genome assemblies, providing a resource for outbreak investigation and research in to population structure, gene association, global epidemiology and vaccine coverage. A Representational State Transfer (REST) Application Programming Interface (API) has been developed for the PubMLST website to make these large quantities of structured molecular typing and whole genome sequence data available for programmatic access by any third party application. The API is an integral component of the Bacterial Isolate Genome Sequence Database (BIGSdb) platform that is used to host PubMLST resources, and exposes all public data within the site. In addition to data browsing, searching and download, the API supports authentication and submission of new data to curator queues. Database URL: http://rest.pubmlst.org/ PMID:29220452

Testing the molecular clock using mechanistic models of fossil preservation and molecular evolution

PubMed Central

2017-01-01

Molecular sequence data provide information about relative times only, and fossil-based age constraints are the ultimate source of information about absolute times in molecular clock dating analyses. Thus, fossil calibrations are critical to molecular clock dating, but competing methods are difficult to evaluate empirically because the true evolutionary time scale is never known. Here, we combine mechanistic models of fossil preservation and sequence evolution in simulations to evaluate different approaches to constructing fossil calibrations and their impact on Bayesian molecular clock dating, and the relative impact of fossil versus molecular sampling. We show that divergence time estimation is impacted by the model of fossil preservation, sampling intensity and tree shape. The addition of sequence data may improve molecular clock estimates, but accuracy and precision is dominated by the quality of the fossil calibrations. Posterior means and medians are poor representatives of true divergence times; posterior intervals provide a much more accurate estimate of divergence times, though they may be wide and often do not have high coverage probability. Our results highlight the importance of increased fossil sampling and improved statistical approaches to generating calibrations, which should incorporate the non-uniform nature of ecological and temporal fossil species distributions. PMID:28637852

MOlecular MAterials Property Prediction Package (MOMAP) 1.0: a software package for predicting the luminescent properties and mobility of organic functional materials

NASA Astrophysics Data System (ADS)

Niu, Yingli; Li, Wenqiang; Peng, Qian; Geng, Hua; Yi, Yuanping; Wang, Linjun; Nan, Guangjun; Wang, Dong; Shuai, Zhigang

2018-04-01

MOlecular MAterials Property Prediction Package (MOMAP) is a software toolkit for molecular materials property prediction. It focuses on luminescent properties and charge mobility properties. This article contains a brief descriptive introduction of key features, theoretical models and algorithms of the software, together with examples that illustrate the performance. First, we present the theoretical models and algorithms for molecular luminescent properties calculation, which includes the excited-state radiative/non-radiative decay rate constant and the optical spectra. Then, a multi-scale simulation approach and its algorithm for the molecular charge mobility are described. This approach is based on hopping model and combines with Kinetic Monte Carlo and molecular dynamics simulations, and it is especially applicable for describing a large category of organic semiconductors, whose inter-molecular electronic coupling is much smaller than intra-molecular charge reorganisation energy.

Dissociation of single-strand DNA: single-walled carbon nanotube hybrids by Watson-Crick base-pairing.

PubMed

Jung, Seungwon; Cha, Misun; Park, Jiyong; Jeong, Namjo; Kim, Gunn; Park, Changwon; Ihm, Jisoon; Lee, Junghoon

2010-08-18

It has been known that single-strand DNA wraps around a single-walled carbon nanotube (SWNT) by pi-stacking. In this paper it is demonstrated that such DNA is dissociated from the SWNT by Watson-Crick base-pairing with a complementary sequence. Measurement of field effect transistor characteristics indicates a shift of the electrical properties as a result of this "unwrapping" event. We further confirm the suggested process through Raman spectroscopy and gel electrophoresis. Experimental results are verified in view of atomistic mechanisms with molecular dynamics simulations and binding energy analyses.

A 90-Kilodalton Endothelial Cell Molecule Mediating Lymphocyte Binding in Humans

NASA Astrophysics Data System (ADS)

Salmi, Marko; Jalkanen, Sirpa

1992-09-01

Interactions between leukocyte surface receptors and their ligands on vascular endothelial cells control lymphocyte traffic between the blood and various lymphoid organs, as well as extravasation of leukocytes into sites of inflammation. A heretofore undescribed 90-kilodalton human endothelial cell adhesion molecule (VAP-1) defined by a monoclonal antibody 1B2 is described. The expression pattern, molecular mass, functional properties, and an amino-terminal amino acid sequence define VAP-1 as an endothelial ligand for lymphocytes. VAP-1 helps to elucidate the complex heterotypic cell interactions that direct tissue-selective lymphocyte migration in man.

Breakpoint structure of the Anopheles gambiae 2Rb chromosomal inversion.

PubMed

Lobo, Neil F; Sangaré, Djibril M; Regier, Allison A; Reidenbach, Kyanne R; Bretz, David A; Sharakhova, Maria V; Emrich, Scott J; Traore, Sekou F; Costantini, Carlo; Besansky, Nora J; Collins, Frank H

2010-10-25

Alternative arrangements of chromosome 2 inversions in Anopheles gambiae are important sources of population structure, and are associated with adaptation to environmental heterogeneity. The forces responsible for their origin and maintenance are incompletely understood. Molecular characterization of inversion breakpoints provides insight into how they arose, and provides the basis for development of molecular karyotyping methods useful in future studies. Sequence comparison of regions near the cytological breakpoints of 2Rb allowed the molecular delineation of breakpoint boundaries. Comparisons were made between the standard 2R+b arrangement in the An. gambiae PEST reference genome and the inverted 2Rb arrangements in the An. gambiae M and S genome assemblies. Sequence differences between alternative 2Rb arrangements were exploited in the design of a PCR diagnostic assay, which was evaluated against the known chromosomal banding pattern of laboratory colonies and field-collected samples from Mali and Cameroon. The breakpoints of the 7.55 Mb 2Rb inversion are flanked by extensive runs of the same short (72 bp) tandemly organized sequence, which was likely responsible for chromosomal breakage and rearrangement. Application of the molecular diagnostic assay suggested that 2Rb has a single common origin in An. gambiae and its sibling species, Anopheles arabiensis, and also that the standard arrangement (2R+b) may have arisen twice through breakpoint reuse. The molecular diagnostic was reliable when applied to laboratory colonies, but its accuracy was lower in natural populations. The complex repetitive sequence flanking the 2Rb breakpoint region may be prone to structural and sequence-level instability. The 2Rb molecular diagnostic has immediate application in studies based on laboratory colonies, but its usefulness in natural populations awaits development of complementary molecular tools.

Design, production and molecular structure of a new family of artificial alpha-helicoidal repeat proteins (αRep) based on thermostable HEAT-like repeats.

PubMed

Urvoas, Agathe; Guellouz, Asma; Valerio-Lepiniec, Marie; Graille, Marc; Durand, Dominique; Desravines, Danielle C; van Tilbeurgh, Herman; Desmadril, Michel; Minard, Philippe

2010-11-26

Repeat proteins have a modular organization and a regular architecture that make them attractive models for design and directed evolution experiments. HEAT repeat proteins, although very common, have not been used as a scaffold for artificial proteins, probably because they are made of long and irregular repeats. Here, we present and validate a consensus sequence for artificial HEAT repeat proteins. The sequence was defined from the structure-based sequence analysis of a thermostable HEAT-like repeat protein. Appropriate sequences were identified for the N- and C-caps. A library of genes coding for artificial proteins based on this sequence design, named αRep, was assembled using new and versatile methodology based on circular amplification. Proteins picked randomly from this library are expressed as soluble proteins. The biophysical properties of proteins with different numbers of repeats and different combinations of side chains in hypervariable positions were characterized. Circular dichroism and differential scanning calorimetry experiments showed that all these proteins are folded cooperatively and are very stable (T(m) >70 °C). Stability of these proteins increases with the number of repeats. Detailed gel filtration and small-angle X-ray scattering studies showed that the purified proteins form either monomers or dimers. The X-ray structure of a stable dimeric variant structure was solved. The protein is folded with a highly regular topology and the repeat structure is organized, as expected, as pairs of alpha helices. In this protein variant, the dimerization interface results directly from the variable surface enriched in aromatic residues located in the randomized positions of the repeats. The dimer was crystallized both in an apo and in a PEG-bound form, revealing a very well defined binding crevice and some structure flexibility at the interface. This fortuitous binding site could later prove to be a useful binding site for other low molecular mass partners. Copyright © 2010 Elsevier Ltd. All rights reserved.

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

PubMed

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

2009-01-01

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

Future collaborations between NEON and the U.S. EPA: linking molecular genomics for bioassessment with national ecological data sets

EPA Science Inventory

Molecular taxonomic techniques such as DNA barcoding offer interesting new capabilities for studying community biodiversity for applications like biological monitoring. Beyond DNA barcoding, new DNA sequencing technologies (i.e. Next-Generation Sequencing) present even greater po...

Stereodivergent synthesis with a programmable molecular machine

NASA Astrophysics Data System (ADS)

Kassem, Salma; Lee, Alan T. L.; Leigh, David A.; Marcos, Vanesa; Palmer, Leoni I.; Pisano, Simone

2017-09-01

It has been convincingly argued that molecular machines that manipulate individual atoms, or highly reactive clusters of atoms, with Ångström precision are unlikely to be realized. However, biological molecular machines routinely position rather less reactive substrates in order to direct chemical reaction sequences, from sequence-specific synthesis by the ribosome to polyketide synthases, where tethered molecules are passed from active site to active site in multi-enzyme complexes. Artificial molecular machines have been developed for tasks that include sequence-specific oligomer synthesis and the switching of product chirality, a photo-responsive host molecule has been described that is able to mechanically twist a bound molecular guest, and molecular fragments have been selectively transported in either direction between sites on a molecular platform through a ratchet mechanism. Here we detail an artificial molecular machine that moves a substrate between different activating sites to achieve different product outcomes from chemical synthesis. This molecular robot can be programmed to stereoselectively produce, in a sequential one-pot operation, an excess of any one of four possible diastereoisomers from the addition of a thiol and an alkene to an α,β-unsaturated aldehyde in a tandem reaction process. The stereodivergent synthesis includes diastereoisomers that cannot be selectively synthesized through conventional iminium-enamine organocatalysis. We anticipate that future generations of programmable molecular machines may have significant roles in chemical synthesis and molecular manufacturing.

Static and Dynamic Properties of DNA Confined in Nanochannels

NASA Astrophysics Data System (ADS)

Gupta, Damini

Next-generation sequencing (NGS) techniques have considerably reduced the cost of high-throughput DNA sequencing. However, it is challenging to detect large-scale genomic variations by NGS due to short read lengths. Genome mapping can easily detect large-scale structural variations because it operates on extremely large intact molecules of DNA with adequate resolution. One of the promising methods of genome mapping is based on confining large DNA molecules inside a nanochannel whose cross-sectional dimensions are approximately 50 nm. Even though this genome mapping technology has been commercialized, the current understanding of the polymer physics of DNA in nanochannel confinement is based on theories and lacks much needed experimental support. The results of this dissertation are aimed at providing a detailed experimental understanding of equilibrium properties of nanochannel-confined DNA molecules. The results are divided into three parts. In first part, we evaluate the role of channel shape on thermodynamic properties of channel confined DNA molecules using a combination of fluorescence microscopy and simulations. Specifically, we show that high aspect ratio of rectangular channels significantly alters the chain statistics as compared to an equivalent square channel with same cross-sectional area. In the second part, we present experimental evidence that weak excluded volume effects arise in DNA nanochannel confinement, which form the physical basis for the extended de Gennes regime. We also show how confinement spectroscopy and simulations can be combined to reduce molecular weight dispersity effects arising from shearing, photo-cleavage, and nonuniform staining of DNA. Finally, the third part of the thesis concerns the dynamic properties of nanochannel confined DNA. We directly measure the center-of-mass diffusivity of single DNA molecules in confinement and show that that it is necessary to modify the classical results of de Gennes to account for local chain stiffness of DNA in order to explain the experimental results. In the end, we believe that our findings from the experimental test of the phase diagram for channel-confined DNA, with careful control over molecular weight dispersity, channel geometry, and electrostatic interactions, will provide a firm foundation for the emerging genome mapping technology.

Molecular Strain Typing of Mycobacterium tuberculosis: a Review of Frequently Used Methods

PubMed Central

2016-01-01

Tuberculosis, caused by the bacterium Mycobacterium tuberculosis, remains one of the most serious global health problems. Molecular typing of M. tuberculosis has been used for various epidemiologic purposes as well as for clinical management. Currently, many techniques are available to type M. tuberculosis. Choosing the most appropriate technique in accordance with the existing laboratory conditions and the specific features of the geographic region is important. Insertion sequence IS6110-based restriction fragment length polymorphism (RFLP) analysis is considered the gold standard for the molecular epidemiologic investigations of tuberculosis. However, other polymerase chain reaction-based methods such as spacer oligonucleotide typing (spoligotyping), which detects 43 spacer sequence-interspersing direct repeats (DRs) in the genomic DR region; mycobacterial interspersed repetitive units–variable number tandem repeats, (MIRU-VNTR), which determines the number and size of tandem repetitive DNA sequences; repetitive-sequence-based PCR (rep-PCR), which provides high-throughput genotypic fingerprinting of multiple Mycobacterium species; and the recently developed genome-based whole genome sequencing methods demonstrate similar discriminatory power and greater convenience. This review focuses on techniques frequently used for the molecular typing of M. tuberculosis and discusses their general aspects and applications. PMID:27709842

Reconstructing genealogies of serial samples under the assumption of a molecular clock using serial-sample UPGMA.

PubMed

Drummond, A; Rodrigo, A G

2000-12-01

Reconstruction of evolutionary relationships from noncontemporaneous molecular samples provides a new challenge for phylogenetic reconstruction methods. With recent biotechnological advances there has been an increase in molecular sequencing throughput, and the potential to obtain serial samples of sequences from populations, including rapidly evolving pathogens, is fast being realized. A new method called the serial-sample unweighted pair grouping method with arithmetic means (sUPGMA) is presented that reconstructs a genealogy or phylogeny of sequences sampled serially in time using a matrix of pairwise distances. The resulting tree depicts the terminal lineages of each sample ending at a different level consistent with the sample's temporal order. Since sUPGMA is a variant of UPGMA, it will perform best when sequences have evolved at a constant rate (i.e., according to a molecular clock). On simulated data, this new method performs better than standard cluster analysis under a variety of longitudinal sampling strategies. Serial-sample UPGMA is particularly useful for analysis of longitudinal samples of viruses and bacteria, as well as ancient DNA samples, with the minimal requirement that samples of sequences be ordered in time.

Molecular Strain Typing of Mycobacterium tuberculosis: a Review of Frequently Used Methods.

PubMed

Ei, Phyu Win; Aung, Wah Wah; Lee, Jong Seok; Choi, Go Eun; Chang, Chulhun L

2016-11-01

Tuberculosis, caused by the bacterium Mycobacterium tuberculosis, remains one of the most serious global health problems. Molecular typing of M. tuberculosis has been used for various epidemiologic purposes as well as for clinical management. Currently, many techniques are available to type M. tuberculosis. Choosing the most appropriate technique in accordance with the existing laboratory conditions and the specific features of the geographic region is important. Insertion sequence IS6110-based restriction fragment length polymorphism (RFLP) analysis is considered the gold standard for the molecular epidemiologic investigations of tuberculosis. However, other polymerase chain reaction-based methods such as spacer oligonucleotide typing (spoligotyping), which detects 43 spacer sequence-interspersing direct repeats (DRs) in the genomic DR region; mycobacterial interspersed repetitive units-variable number tandem repeats, (MIRU-VNTR), which determines the number and size of tandem repetitive DNA sequences; repetitive-sequence-based PCR (rep-PCR), which provides high-throughput genotypic fingerprinting of multiple Mycobacterium species; and the recently developed genome-based whole genome sequencing methods demonstrate similar discriminatory power and greater convenience. This review focuses on techniques frequently used for the molecular typing of M. tuberculosis and discusses their general aspects and applications.

A critical re-evaluation of multilocus sequence typing (MLST) efforts in Wolbachia.

PubMed

Bleidorn, Christoph; Gerth, Michael

2018-01-01

Wolbachia (Alphaproteobacteria, Rickettsiales) is the most common, and arguably one of the most important inherited symbionts. Molecular differentiation of Wolbachia strains is routinely performed with a set of five multilocus sequence typing (MLST) markers. However, since its inception in 2006, the performance of MLST in Wolbachia strain typing has not been assessed objectively. Here, we evaluate the properties of Wolbachia MLST markers and compare it to 252 other single copy loci present in the genome of most Wolbachia strains. Specifically, we investigated how well MLST performs at strain differentiation, at reflecting genetic diversity of strains, and as phylogenetic marker. We find that MLST loci are outperformed by other loci at all tasks they are currently employed for, and thus that they do not reflect the properties of a Wolbachia strain very well. We argue that whole genome typing approaches should be used for Wolbachia typing in the future. Alternatively, if few loci approaches are necessary, we provide a characterisation of 252 single copy loci for a number a criteria, which may assist in designing specific typing systems or phylogenetic studies. © FEMS 2017. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Relative stability of major types of beta-turns as a function of amino acid composition: a study based on Ab initio energetic and natural abundance data.

PubMed

Perczel, András; Jákli, Imre; McAllister, Michael A; Csizmadia, Imre G

2003-06-06

Folding properties of small globular proteins are determined by their amino acid sequence (primary structure). This holds both for local (secondary structure) and for global conformational features of linear polypeptides and proteins composed from natural amino acid derivatives. It thus provides the rational basis of structure prediction algorithms. The shortest secondary structure element, the beta-turn, most typically adopts either a type I or a type II form, depending on the amino acid composition. Herein we investigate the sequence-dependent folding stability of both major types of beta-turns using simple dipeptide models (-Xxx-Yyy-). Gas-phase ab initio properties of 16 carefully selected and suitably protected dipeptide models (for example Val-Ser, Ala-Gly, Ser-Ser) were studied. For each backbone fold most probable side-chain conformers were considered. Fully optimized 321G RHF molecular structures were employed in medium level [B3LYP/6-311++G(d,p)//RHF/3-21G] energy calculations to estimate relative populations of the different backbone conformers. Our results show that the preference for beta-turn forms as calculated by quantum mechanics and observed in Xray determined proteins correlates significantly.

Identification of Arcanobacterium pyogenes isolated by post mortem examinations of a bearded dragon and a gecko by phenotypic and genotypic properties

PubMed Central

Ülbegi-Mohyla, H.; Hijazin, M.; Alber, J.; Hassan, A. A.; Abdulmawjood, A.; Prenger-Berninghoff, E.; Weiß, R.; Zschöck, M.

2010-01-01

The present study was designed to identify phenotypically and genotypically two Arcanobacterium (A.) pyogenes strains isolated by post mortem examinations of a bearded dragon and a gecko. The A. pyogenes strains showed the typical biochemical properties and displayed CAMP-like synergistic hemolytic activities with various indicator strains. The species identity could be confirmed genotypically by amplification and sequencing of the 16S rDNA gene and, as novel target gene, by sequencing of the beta subunit of RNA polymerase encoding gene rpoB, of both strains and of reference strains representing nine species of the genus Arcanobacterium. The species identity of the two A. pyogenes strains could additionally be confirmed by PCR mediated amplification of species specific parts of the 16S-23S rDNA intergenic spacer region, the pyolysin encoding gene plo and by amplification of the collagen-binding protein encoding gene cbpA. All these molecular targets might help to improve the future identification and further characterization of A. pyogenes which, as demonstrated in the present study, could also be isolated from reptile specimens. PMID:20706035

Do recent US Supreme Court rulings on patenting of genes and genetic diagnostics affect the practice of genetic screening and diagnosis in prenatal and reproductive care?

PubMed Central

Chandrasekharan, Subhashini; McGuire, Amy L.; Van den Veyver, Ignatia B.

2015-01-01

Thousands of patents have been awarded that claim human gene sequences and their uses, and some have been challenged in court. In a recent high-profile case, Association for Molecular Pathology, et al. vs. Myriad Genetics, Inc., et al., the United States Supreme Court ruled that genes are natural occurring substances and therefore not patentable through “composition of matter” claims. The consequences of this ruling will extend well beyond ending Myriad's monopoly over BRCA testing, and may affect similar monopolies of other commercial laboratories for tests involving other genes. It could also simplify intellectual property issues surrounding genome-wide clinical sequencing, which can generate results for genes covered by intellectual property. Non-invasive prenatal testing (NIPT) for common aneuploidies using cell-free fetal (cff) DNA in maternal blood is currently offered through commercial laboratories and is also the subject of ongoing patent litigation. The recent Supreme Court decision in the Myriad case has already been invoked by a lower district court in NIPT litigation and resulted in invalidation of primary claims in a patent on currently marketed cffDNA-based testing for chromosomal aneuploidies. PMID:24989832

The Origin and Early Evolution of Membrane Proteins

NASA Technical Reports Server (NTRS)

Pohorille, Andrew; Schweighofter, Karl; Wilson, Michael A.

2006-01-01

The origin and early evolution of membrane proteins, and in particular ion channels, are considered from the point of view that the transmembrane segments of membrane proteins are structurally quite simple and do not require specific sequences to fold. We argue that the transport of solute species, especially ions, required an early evolution of efficient transport mechanisms, and that the emergence of simple ion channels was protobiologically plausible. We also argue that, despite their simple structure, such channels could possess properties that, at the first sight, appear to require markedly larger complexity. These properties can be subtly modulated by local modifications to the sequence rather than global changes in molecular architecture. In order to address the evolution and development of ion channels, we focus on identifying those protein domains that are commonly associated with ion channel proteins and are conserved throughout the three main domains of life (Eukarya, Prokarya, and Archaea). We discuss the potassium-sodium-calcium superfamily of voltage-gated ion channels, mechanosensitive channels, porins, and ABC-transporters and argue that these families of membrane channels have sufficiently universal architectures that they can readily adapt to the diverse functional demands arising during evolution.

Isolation and characterization of two serine proteases from metagenomic libraries of the Gobi and Death Valley deserts.

PubMed

Neveu, Julie; Regeard, Christophe; DuBow, Michael S

2011-08-01

The screening of environmental DNA metagenome libraries for functional activities can provide an important source of new molecules and enzymes. In this study, we identified 17 potential protease-producing clones from two metagenomic libraries derived from samples of surface sand from the Gobi and Death Valley deserts. Two of the proteases, DV1 and M30, were purified and biochemically examined. These two proteases displayed a molecular mass of 41.5 kDa and 45.7 kDa, respectively, on SDS polyacrylamide gels. Alignments with known protease sequences showed less than 55% amino acid sequence identity. These two serine proteases appear to belong to the subtilisin (S8A) family and displayed several unique biochemical properties. Protease DV1 had an optimum pH of 8 and an optimal activity at 55°C, while protease M30 had an optimum pH >11 and optimal activity at 40°C. The properties of these enzymes make them potentially useful for biotechnological applications and again demonstrate that metagenomic approaches can be useful, especially when coupled with the study of novel environments such as deserts.

From protein sequence to dynamics and disorder with DynaMine.

PubMed

Cilia, Elisa; Pancsa, Rita; Tompa, Peter; Lenaerts, Tom; Vranken, Wim F

2013-01-01

Protein function and dynamics are closely related; however, accurate dynamics information is difficult to obtain. Here based on a carefully assembled data set derived from experimental data for proteins in solution, we quantify backbone dynamics properties on the amino-acid level and develop DynaMine--a fast, high-quality predictor of protein backbone dynamics. DynaMine uses only protein sequence information as input and shows great potential in distinguishing regions of different structural organization, such as folded domains, disordered linkers, molten globules and pre-structured binding motifs of different sizes. It also identifies disordered regions within proteins with an accuracy comparable to the most sophisticated existing predictors, without depending on prior disorder knowledge or three-dimensional structural information. DynaMine provides molecular biologists with an important new method that grasps the dynamical characteristics of any protein of interest, as we show here for human p53 and E1A from human adenovirus 5.

Structural and functional characterization of a calcium-activated cation channel from Tsukamurella paurometabola

NASA Astrophysics Data System (ADS)

Dhakshnamoorthy, Balasundaresan; Rohaim, Ahmed; Rui, Huan; Blachowicz, Lydia; Roux, Benoît

2016-09-01

The selectivity filter is an essential functional element of K+ channels that is highly conserved both in terms of its primary sequence and its three-dimensional structure. Here, we investigate the properties of an ion channel from the Gram-positive bacterium Tsukamurella paurometabola with a selectivity filter formed by an uncommon proline-rich sequence. Electrophysiological recordings show that it is a non-selective cation channel and that its activity depends on Ca2+ concentration. In the crystal structure, the selectivity filter adopts a novel conformation with Ca2+ ions bound within the filter near the pore helix where they are coordinated by backbone oxygen atoms, a recurrent motif found in multiple proteins. The binding of Ca2+ ion in the selectivity filter controls the widening of the pore as shown in crystal structures and in molecular dynamics simulations. The structural, functional and computational data provide a characterization of this calcium-gated cationic channel.

Applications of Mass Spectrometry to Structural Analysis of Marine Oligosaccharides

PubMed Central

Lang, Yinzhi; Zhao, Xia; Liu, Lili; Yu, Guangli

2014-01-01

Marine oligosaccharides have attracted increasing attention recently in developing potential drugs and biomaterials for their particular physical and chemical properties. However, the composition and sequence analysis of marine oligosaccharides are very challenging for their structural complexity and heterogeneity. Mass spectrometry (MS) has become an important technique for carbohydrate analysis by providing more detailed structural information, including molecular mass, sugar constituent, sequence, inter-residue linkage position and substitution pattern. This paper provides an overview of the structural analysis based on MS approaches in marine oligosaccharides, which are derived from some biologically important marine polysaccharides, including agaran, carrageenan, alginate, sulfated fucan, chitosan, glycosaminoglycan (GAG) and GAG-like polysaccharides. Applications of electrospray ionization mass spectrometry (ESI-MS) are mainly presented and the general applications of matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) are also outlined. Some technical challenges in the structural analysis of marine oligosaccharides by MS have also been pointed out. PMID:24983643

Mesomorphic phase transitions of 3F7HPhF studied by complementary methods

NASA Astrophysics Data System (ADS)

Deptuch, Aleksandra; Jaworska-Gołąb, Teresa; Marzec, Monika; Pociecha, Damian; Fitas, Jakub; Żurowska, Magdalena; Tykarska, Marzena; Hooper, James

2018-02-01

Physical properties and the phase sequence of (S)-4‧-(1-methylheptyloxycarbonyl)biphenyl-4-yl 4-[7-(2,2,3,3,4,4,4-heptafluorobutoxy) heptyl-1-oxy]-2-fluorobenzoate exhibiting the liquid crystalline paraelectric smectic A*, ferroelectric smectic C* and antiferroelectric smectic CA* phases were studied by complementary methods in the temperature range from -125 to 120 °C. Differential scanning calorimetry measurements together with polarizing optical microscopy provided the phase sequence, including the glass transition and a cold crystallization. X-ray diffraction was used to obtain the unit-cell parameters of the crystal phase, as well as the layer thickness and correlation length in the liquid crystalline smectic phases. The tilt angle was found to reach 45°, as determined from the measurements of the layer thickness and molecular modeling. Relaxation processes in the smectic phases and the fragility parameter were studied using frequency-domain dielectric spectroscopy.

Sequence-dependent DNA flexibility mediates DNase I cleavage.

PubMed

Heddi, Brahim; Abi-Ghanem, Josephine; Lavigne, Marc; Hartmann, Brigitte

2010-01-08

Understanding the preference of nonspecific proteins for certain DNA structural features requires an accurate description of the properties of free DNA, especially regarding their possible predisposition to adopt a conformation that favors the formation of a complex. Exploiting previous exhaustive NMR studies performed on free DNA oligomers, we investigated the molecular basis of DNase I sensitivity under conditions where DNase I binding limits the probability of cleavage. We showed that cleavage intensity was correlated with adjacent 3' phosphate linkage flexibility, monitored by (31)P chemical shifts. Examining NMR-refined DNA structures highlighted that sequence-dependent flexible phosphates were associated with large minor groove variations that may promote the affinity of DNase I, according to relevant DNA-protein complexes. In sum, this work demonstrates that specificity in DNA-DNase I interaction is mediated by DNA flexibility, which influences the induced-fit transitions required to form productive complexes.

Transcriptome Analysis and Development of SSR Molecular Markers in Glycyrrhiza uralensis Fisch.

PubMed Central

Liu, Yaling; Zhang, Pengfei; Song, Meiling; Hou, Junling; Qing, Mei; Wang, Wenquan; Liu, Chunsheng

2015-01-01

Licorice is an important traditional Chinese medicine with clinical and industrial applications. Genetic resources of licorice are insufficient for analysis of molecular biology and genetic functions; as such, transcriptome sequencing must be conducted for functional characterization and development of molecular markers. In this study, transcriptome sequencing on the Illumina HiSeq 2500 sequencing platform generated a total of 5.41 Gb clean data. De novo assembly yielded a total of 46,641 unigenes. Comparison analysis using BLAST showed that the annotations of 29,614 unigenes were conserved. Further study revealed 773 genes related to biosynthesis of secondary metabolites of licorice, 40 genes involved in biosynthesis of the terpenoid backbone, and 16 genes associated with biosynthesis of glycyrrhizic acid. Analysis of unigenes larger than 1 Kb with a length of 11,702 nt presented 7,032 simple sequence repeats (SSR). Sixty-four of 69 randomly designed and synthesized SSR pairs were successfully amplified, 33 pairs of primers were polymorphism in in Glycyrrhiza uralensis Fisch., Glycyrrhiza inflata Bat., Glycyrrhiza glabra L. and Glycyrrhiza pallidiflora Maxim. This study not only presents the molecular biology data of licorice but also provides a basis for genetic diversity research and molecular marker-assisted breeding of licorice. PMID:26571372

A Study of Two Instructional Sequences Informed by Alternative Learning Progressions in Genetics

NASA Astrophysics Data System (ADS)

Duncan, Ravit Golan; Choi, Jinnie; Castro-Faix, Moraima; Cavera, Veronica L.

2017-12-01

Learning progressions (LPs) are hypothetical models of how learning in a domain develops over time with appropriate instruction. In the domain of genetics, there are two independently developed alternative LPs. The main difference between the two progressions hinges on their assumptions regarding the accessibility of classical (Mendelian) versus molecular genetics and the order in which they should be taught. In order to determine the relative difficulty of the different genetic ideas included in the two progressions, and to test which one is a better fit with students' actual learning, we developed two modules in classical and molecular genetics and alternated their sequence in an implementation study with 11th grade students studying biology. We developed a set of 56 ordered multiple-choice items that collectively assessed both molecular and classical genetic ideas. We found significant gains in students' learning in both molecular and classical genetics, with the largest gain relating to understanding the informational content of genes and the smallest gain in understanding modes of inheritance. Using multidimensional item response modeling, we found no statistically significant differences between the two instructional sequences. However, there was a trend of slightly higher gains for the molecular-first sequence for all genetic ideas.

Molecular Findings Among Patients Referred for Clinical Whole-Exome Sequencing

PubMed Central

Yang, Yaping; Muzny, Donna M.; Xia, Fan; Niu, Zhiyv; Person, Richard; Ding, Yan; Ward, Patricia; Braxton, Alicia; Wang, Min; Buhay, Christian; Veeraraghavan, Narayanan; Hawes, Alicia; Chiang, Theodore; Leduc, Magalie; Beuten, Joke; Zhang, Jing; He, Weimin; Scull, Jennifer; Willis, Alecia; Landsverk, Megan; Craigen, William J.; Bekheirnia, Mir Reza; Stray-Pedersen, Asbjorg; Liu, Pengfei; Wen, Shu; Alcaraz, Wendy; Cui, Hong; Walkiewicz, Magdalena; Reid, Jeffrey; Bainbridge, Matthew; Patel, Ankita; Boerwinkle, Eric; Beaudet, Arthur L.; Lupski, James R.; Plon, Sharon E.; Gibbs, Richard A.; Eng, Christine M.

2015-01-01

IMPORTANCE Clinical whole-exome sequencing is increasingly used for diagnostic evaluation of patients with suspected genetic disorders. OBJECTIVE To perform clinical whole-exome sequencing and report (1) the rate of molecular diagnosis among phenotypic groups, (2) the spectrum of genetic alterations contributing to disease, and (3) the prevalence of medically actionable incidental findings such as FBN1 mutations causing Marfan syndrome. DESIGN, SETTING, AND PATIENTS Observational study of 2000 consecutive patients with clinical whole-exome sequencing analyzed between June 2012 and August 2014. Whole-exome sequencing tests were performed at a clinical genetics laboratory in the United States. Results were reported by clinical molecular geneticists certified by the American Board of Medical Genetics and Genomics. Tests were ordered by the patient’s physician. The patients were primarily pediatric (1756 [88%]; mean age, 6 years; 888 females [44%], 1101 males [55%], and 11 fetuses [1% gender unknown]), demonstrating diverse clinical manifestations most often including nervous system dysfunction such as developmental delay. MAIN OUTCOMES AND MEASURES Whole-exome sequencing diagnosis rate overall and by phenotypic category, mode of inheritance, spectrum of genetic events, and reporting of incidental findings. RESULTS A molecular diagnosis was reported for 504 patients (25.2%) with 58% of the diagnostic mutations not previously reported. Molecular diagnosis rates for each phenotypic category were 143/526 (27.2%; 95% CI, 23.5%–31.2%) for the neurological group, 282/1147 (24.6%; 95% CI, 22.1%–27.2%) for the neurological plus other organ systems group, 30/83 (36.1%; 95% CI, 26.1%–47.5%) for the specific neurological group, and 49/244 (20.1%; 95% CI, 15.6%–25.8%) for the nonneurological group. The Mendelian disease patterns of the 527 molecular diagnoses included 280 (53.1%) autosomal dominant, 181 (34.3%) autosomal recessive (including 5 with uniparental disomy), 65 (12.3%) X-linked, and 1 (0.2%) mitochondrial. Of 504 patients with a molecular diagnosis, 23 (4.6%) had blended phenotypes resulting from 2 single gene defects. About 30% of the positive cases harbored mutations in disease genes reported since 2011. There were 95 medically actionable incidental findings in genes unrelated to the phenotype but with immediate implications for management in 92 patients (4.6%), including 59 patients (3%) with mutations in genes recommended for reporting by the American College of Medical Genetics and Genomics. CONCLUSIONS AND RELEVANCE Whole-exome sequencing provided a potential molecular diagnosis for 25% of a large cohort of patients referred for evaluation of suspected genetic conditions, including detection of rare genetic events and new mutations contributing to disease. The yield of whole-exome sequencing may offer advantages over traditional molecular diagnostic approaches in certain patients. PMID:25326635

Identification and in silico characterization of a novel peptide inhibitor of angiotensin converting enzyme from pigeon pea (Cajanus cajan).

PubMed

Nawaz, K A Ayub; David, Swapna Merlin; Murugesh, Easwaran; Thandeeswaran, Murugesan; Kiran, Kalarikkal Gopikrishnan; Mahendran, Ramasamy; Palaniswamy, Muthusamy; Angayarkanni, Jayaraman

2017-12-01

Plants are important sources of bioactive peptides. Among these, angiotensin converting enzyme (ACE) inhibitory peptides have a major focus on their ability to prevent hypertension. Inhibition of ACE has been established as an effective approach for the treatment of ACE associated diseases. Some synthetic ACE inhibitory drugs cause side effects and hence there is a constant interest in natural compounds as alternatives. The study was designed to identify and characterize a peptide molecule from pigeon pea which has the biological property to inhibit ACE and can be developed as a therapeutic approach towards hypertension. Seeds of pigeon pea (Cajanus cajan (L.) Millsp.) was fermented with Aspergillus niger, a proteolytic fungus isolated from spoiled milk sweet. The extract was purified by size exclusion chromatography by FPLC system. The fractions that showed ACE inhibition was subjected to LC-MS/MS for sequence identification. The stability of the peptide was analyzed by molecular dynamic simulations and the interaction sites with ACE were identified by molecular docking. The study report a novel ACE inhibitory octapeptide Val-Val-Ser-Leu-Ser-Ile-Pro-Arg with a molecular mass of 869.53 Da. The Lineweaver-Burk plot indicated that the inhibition of ACE by this peptide is in competitive mode. Also, molecular docking and simulation studies showed a strong and stable interaction of the peptide with ACE. The results clearly show the inhibitory property of the peptide against ACE and hence it can be explored as a therapeutic strategy towards hypertension and other ACE associated diseases. Copyright © 2017 Elsevier GmbH. All rights reserved.

A search for pre-main sequence stars in the high-latitude molecular clouds. II - A survey of the Einstein database

NASA Technical Reports Server (NTRS)

Caillault, Jean-Pierre; Magnani, Loris

1990-01-01

The preliminary results are reported of a survey of every EINSTEIN image which overlaps any high-latitude molecular cloud in a search for X-ray emitting pre-main sequence stars. This survey, together with complementary KPNO and IRAS data, will allow the determination of how prevalent low mass star formation is in these clouds in general and, particularly, in the translucent molecular clouds.

Functional Testing of SLC26A4 Variants—Clinical and Molecular Analysis of a Cohort with Enlarged Vestibular Aqueduct from Austria

PubMed Central

Bernardinelli, Emanuele; Nofziger, Charity; Patsch, Wolfgang; Rasp, Gerd; Paulmichl, Markus; Dossena, Silvia

2018-01-01

The prevalence and spectrum of sequence alterations in the SLC26A4 gene, which codes for the anion exchanger pendrin, are population-specific and account for at least 50% of cases of non-syndromic hearing loss associated with an enlarged vestibular aqueduct. A cohort of nineteen patients from Austria with hearing loss and a radiological alteration of the vestibular aqueduct underwent Sanger sequencing of SLC26A4 and GJB2, coding for connexin 26. The pathogenicity of sequence alterations detected was assessed by determining ion transport and molecular features of the corresponding SLC26A4 protein variants. In this group, four uncharacterized sequence alterations within the SLC26A4 coding region were found. Three of these lead to protein variants with abnormal functional and molecular features, while one should be considered with no pathogenic potential. Pathogenic SLC26A4 sequence alterations were only found in 12% of patients. SLC26A4 sequence alterations commonly found in other Caucasian populations were not detected. This survey represents the first study on the prevalence and spectrum of SLC26A4 sequence alterations in an Austrian cohort and further suggests that genetic testing should always be integrated with functional characterization and determination of the molecular features of protein variants in order to unequivocally identify or exclude a causal link between genotype and phenotype. PMID:29320412

Improved growth of GaN layers on ultra thin silicon nitride/Si (1 1 1) by RF-MBE

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

Kumar, Mahesh; Roul, Basanta; Central Research Laboratory, Bharat Electronics, Bangalore 560013

High-quality GaN epilayers were grown on Si (1 1 1) substrates by molecular beam epitaxy using a new growth process sequence which involved a substrate nitridation at low temperatures, annealing at high temperatures, followed by nitridation at high temperatures, deposition of a low-temperature buffer layer, and a high-temperature overgrowth. The material quality of the GaN films was also investigated as a function of nitridation time and temperature. Crystallinity and surface roughness of GaN was found to improve when the Si substrate was treated under the new growth process sequence. Micro-Raman and photoluminescence (PL) measurement results indicate that the GaN filmmore » grown by the new process sequence has less tensile stress and optically good. The surface and interface structures of an ultra thin silicon nitride film grown on the Si surface are investigated by core-level photoelectron spectroscopy and it clearly indicates that the quality of silicon nitride notably affects the properties of GaN growth.« less

Complete genome sequence of Lactobacillus plantarum LZ206, a potential probiotic strain with antimicrobial activity against food-borne pathogenic microorganisms.

PubMed

Li, Ping; Gu, Qing; Zhou, Qingqing

2016-11-20

Lactobacilli strains have been considered as important candidates for manufacturing "natural food", due to their antimicrobial properties and generally regarded as safe (GRAS) status. Lactobacillus plantarum LZ206 is a potential probiotic strain isolated from raw cow milk, with antimicrobial activity against various pathogens, including Gram-positive bacteria (Staphylococcus aureus and Listeria monocytogenes), Gram-negtive bacteria (Escherichia coli and Salmonella enterica), and fungus Candida albicans. To better understand molecular base for its antimicrobial activity, entire genome of LZ206 was sequenced. It was revealed that genome of LZ206 contained a circular 3,212,951-bp chromosome, two circular plasmids and one predicted linear plasmid. A plantaricin gene cluster, which is responsible for bacteriocins biosynthesis and could be associated with its broad-spectrum antimicrobial activity, was identified based on comparative genomic analysis. Whole genome sequencing of L. plantarum LZ206 might facilitate its applications to protect food products from pathogens' contamination in the dairy industry. Copyright © 2016 Elsevier B.V. All rights reserved.

Profiling the transcriptome of Gracilaria changii (Rhodophyta) in response to light deprivation.

PubMed

Ho, Chai-Ling; Teoh, Seddon; Teo, Swee-Sen; Rahim, Raha Abdul; Phang, Siew-Moi

2009-01-01

Light regulates photosynthesis, growth and reproduction, yield and properties of phycocolloids, and starch contents in seaweeds. Despite its importance as an environmental cue that regulates many developmental, physiological, and biochemical processes, the network of genes involved during light deprivation are obscure. In this study, we profiled the transcriptome of Gracilaria changii at two different irradiance levels using a cDNA microarray containing more than 3,000 cDNA probes. Microarray analysis revealed that 93 and 105 genes were up- and down-regulated more than 3-fold under light deprivation, respectively. However, only 50% of the transcripts have significant matches to the nonredundant peptide sequences in the database. The transcripts that accumulated under light deprivation include vanadium chloroperoxidase, thioredoxin, ferredoxin component, and reduced nicotinamide adenine dinucleotide dehydrogenase. Among the genes that were down-regulated under light deprivation were genes encoding light harvesting protein, light harvesting complex I, phycobilisome 7.8 kDa linker polypeptide, low molecular weight early light-inducible protein, and vanadium bromoperoxidase. Our findings also provided important clues to the functions of many unknown sequences that could not be annotated using sequence comparison.

Corynebacterium pilbarense sp. nov., a non-lipophilic corynebacterium isolated from a human ankle aspirate.

PubMed

Aravena-Roman, M; Spröer, C; Sträubler, B; Inglis, T; Yassin, A F

2010-07-01

A non-lipophilic coryneform bacterium isolated from an anaerobic Bactec bottle inoculated with an ankle aspirate from a male patient was characterized by phenotypic and molecular taxonomic methods. Chemotaxonomic investigations revealed the presence of short-chain mycolic acids in the cell wall of the bacterium, a feature consistent with members of the genus Corynebacterium. Comparative 16S rRNA gene sequence analysis demonstrated that the isolate displayed 92.0-99.0 % gene sequence similarity with members of the genus Corynebacterium, with Corynebacterium ureicelerivorans as the most closely related phylogenetic species (99.0 % gene sequence similarity). However, the isolate could be genomically separated from C. ureicelerivorans on the basis of DNA-DNA hybridization studies (39.5 % relatedness). Furthermore, the isolate could also be differentiated from C. ureicelerivorans and other species of the genus Corynebacterium on the basis of biochemical properties. Based on both phenotypic and phylogenetic evidence, it is proposed that this isolate be classified as representing a novel species, Corynebacterium pilbarense sp. nov. (type strain IMMIB WACC 658(T)=DSM 45350(T)=CCUG 57942(T)).

Mumps virus F gene and HN gene sequencing as a molecular tool to study mumps virus transmission.

PubMed

Gouma, Sigrid; Cremer, Jeroen; Parkkali, Saara; Veldhuijzen, Irene; van Binnendijk, Rob S; Koopmans, Marion P G

2016-11-01

Various mumps outbreaks have occurred in the Netherlands since 2004, particularly among persons who had received 2 doses of measles, mumps, and rubella (MMR) vaccination. Genomic typing of pathogens can be used to track outbreaks, but the established genotyping of mumps virus based on the small hydrophobic (SH) gene sequences did not provide sufficient resolution. Therefore, we expanded the sequencing to include fusion (F) gene and haemagglutinin-neuraminidase (HN) gene sequences in addition to the SH gene sequences from 109 mumps virus genotype G strains obtained between 2004 and mid 2015 in the Netherlands. When the molecular information from these 3 genes was combined, we were able to identify separate mumps virus clusters and track mumps virus transmission. The analyses suggested that multiple mumps virus introductions occurred in the Netherlands between 2004 and 2015 resulting in several mumps outbreaks throughout this period, whereas during some local outbreaks the molecular data pointed towards endemic circulation. Combined analysis of epidemiological data and sequence data collected in 2015 showed good support for the phylogenetic clustering. Copyright © 2016 Elsevier B.V. All rights reserved.

Using next-generation sequencing to develop molecular diagnostics for Pseudoperonospora cubensis, the cucurbit downy mildew pathogen

USDA-ARS?s Scientific Manuscript database

Advances in Next Generation Sequencing (NGS) allow for rapid development of genomics resources needed to generate molecular diagnostics assays for infectious agents. NGS approaches are particularly helpful for organisms that cannot be cultured, such as the downy mildew pathogens, a group of biotrop...

INVESTIGATIONS INTO MOLECULAR PATHWAYS IN THE POST GENOME ERA: CROSS SPECIES COMPARATIVE GENOMICS APPROACH

EPA Science Inventory

Genome sequencing efforts in the past decade were aimed at generating draft sequences of many prokaryotic and eukaryotic model organisms. Successful completion of unicellular eukaryotes, worm, fly and human genome have opened up the new field of molecular biology and function...

Phylogenetic stratigraphy in the Guerrero Negro hypersaline microbial mat.

PubMed

Harris, J Kirk; Caporaso, J Gregory; Walker, Jeffrey J; Spear, John R; Gold, Nicholas J; Robertson, Charles E; Hugenholtz, Philip; Goodrich, Julia; McDonald, Daniel; Knights, Dan; Marshall, Paul; Tufo, Henry; Knight, Rob; Pace, Norman R

2013-01-01

The microbial mats of Guerrero Negro (GN), Baja California Sur, Mexico historically were considered a simple environment, dominated by cyanobacteria and sulfate-reducing bacteria. Culture-independent rRNA community profiling instead revealed these microbial mats as among the most phylogenetically diverse environments known. A preliminary molecular survey of the GN mat based on only ∼1500 small subunit rRNA gene sequences discovered several new phylum-level groups in the bacterial phylogenetic domain and many previously undetected lower-level taxa. We determined an additional ∼119,000 nearly full-length sequences and 28,000 >200 nucleotide 454 reads from a 10-layer depth profile of the GN mat. With this unprecedented coverage of long sequences from one environment, we confirm the mat is phylogenetically stratified, presumably corresponding to light and geochemical gradients throughout the depth of the mat. Previous shotgun metagenomic data from the same depth profile show the same stratified pattern and suggest that metagenome properties may be predictable from rRNA gene sequences. We verify previously identified novel lineages and identify new phylogenetic diversity at lower taxonomic levels, for example, thousands of operational taxonomic units at the family-genus levels differ considerably from known sequences. The new sequences populate parts of the bacterial phylogenetic tree that previously were poorly described, but indicate that any comprehensive survey of GN diversity has only begun. Finally, we show that taxonomic conclusions are generally congruent between Sanger and 454 sequencing technologies, with the taxonomic resolution achieved dependent on the abundance of reference sequences in the relevant region of the rRNA tree of life.

Integrated analysis of 454 and Illumina transcriptomic sequencing characterizes carbon flux and energy source for fatty acid synthesis in developing Lindera glauca fruits for woody biodiesel.

PubMed

Lin, Zixin; An, Jiyong; Wang, Jia; Niu, Jun; Ma, Chao; Wang, Libing; Yuan, Guanshen; Shi, Lingling; Liu, Lili; Zhang, Jinsong; Zhang, Zhixiang; Qi, Ji; Lin, Shanzhi

2017-01-01

Lindera glauca fruit with high quality and quantity of oil has emerged as a novel potential source of biodiesel in China, but the molecular regulatory mechanism of carbon flux and energy source for oil biosynthesis in developing fruits is still unknown. To better develop fruit oils of L. glauca as woody biodiesel, a combination of two different sequencing platforms (454 and Illumina) and qRT-PCR analysis was used to define a minimal reference transcriptome of developing L. glauca fruits, and to construct carbon and energy metabolic model for regulation of carbon partitioning and energy supply for FA biosynthesis and oil accumulation. We first analyzed the dynamic patterns of growth tendency, oil content, FA compositions, biodiesel properties, and the contents of ATP and pyridine nucleotide of L. glauca fruits from seven different developing stages. Comprehensive characterization of transcriptome of the developing L. glauca fruit was performed using a combination of two different next-generation sequencing platforms, of which three representative fruit samples (50, 125, and 150 DAF) and one mixed sample from seven developing stages were selected for Illumina and 454 sequencing, respectively. The unigenes separately obtained from long and short reads (201, and 259, respectively, in total) were reconciled using TGICL software, resulting in a total of 60,031 unigenes (mean length = 1061.95 bp) to describe a transcriptome for developing L. glauca fruits. Notably, 198 genes were annotated for photosynthesis, sucrose cleavage, carbon allocation, metabolite transport, acetyl-CoA formation, oil synthesis, and energy metabolism, among which some specific transporters, transcription factors, and enzymes were identified to be implicated in carbon partitioning and energy source for oil synthesis by an integrated analysis of transcriptomic sequencing and qRT-PCR. Importantly, the carbon and energy metabolic model was well established for oil biosynthesis of developing L. glauca fruits, which could help to reveal the molecular regulatory mechanism of the increased oil production in developing fruits. This study presents for the first time the application of an integrated two different sequencing analyses (Illumina and 454) and qRT-PCR detection to define a minimal reference transcriptome for developing L. glauca fruits, and to elucidate the molecular regulatory mechanism of carbon flux control and energy provision for oil synthesis. Our results will provide a valuable resource for future fundamental and applied research on the woody biodiesel plants.

A comprehensively molecular haplotype-resolved genome of a European individual

PubMed Central

Suk, Eun-Kyung; McEwen, Gayle K.; Duitama, Jorge; Nowick, Katja; Schulz, Sabrina; Palczewski, Stefanie; Schreiber, Stefan; Holloway, Dustin T.; McLaughlin, Stephen; Peckham, Heather; Lee, Clarence; Huebsch, Thomas; Hoehe, Margret R.

2011-01-01

Independent determination of both haplotype sequences of an individual genome is essential to relate genetic variation to genome function, phenotype, and disease. To address the importance of phase, we have generated the most complete haplotype-resolved genome to date, “Max Planck One” (MP1), by fosmid pool-based next generation sequencing. Virtually all SNPs (>99%) and 80,000 indels were phased into haploid sequences of up to 6.3 Mb (N50 ∼1 Mb). The completeness of phasing allowed determination of the concrete molecular haplotype pairs for the vast majority of genes (81%) including potential regulatory sequences, of which >90% were found to be constituted by two different molecular forms. A subset of 159 genes with potentially severe mutations in either cis or trans configurations exemplified in particular the role of phase for gene function, disease, and clinical interpretation of personal genomes (e.g., BRCA1). Extended genomic regions harboring manifold combinations of physically and/or functionally related genes and regulatory elements were resolved into their underlying “haploid landscapes,” which may define the functional genome. Moreover, the majority of genes and functional sequences were found to contain individual or rare SNPs, which cannot be phased from population data alone, emphasizing the importance of molecular phasing for characterizing a genome in its molecular individuality. Our work provides the foundation to understand that the distinction of molecular haplotypes is essential to resolve the (inherently individual) biology of genes, genomes, and disease, establishing a reference point for “phase-sensitive” personal genomics. MP1's annotated haploid genomes are available as a public resource. PMID:21813624

On the auto and cross correlation of PN sequences

NASA Technical Reports Server (NTRS)

Morakis, J. C.

1969-01-01

The autocorrelation and crosscorrelation properties of pseudorandom (PN) sequences are analyzed by using some important properties of PN sequences. These properties make this discussion understandable without the need of linear algebraic approach. The analysis is followed by some experimental results.

Molecular Phylogenetics: Concepts for a Newcomer.

PubMed

Ajawatanawong, Pravech

Molecular phylogenetics is the study of evolutionary relationships among organisms using molecular sequence data. The aim of this review is to introduce the important terminology and general concepts of tree reconstruction to biologists who lack a strong background in the field of molecular evolution. Some modern phylogenetic programs are easy to use because of their user-friendly interfaces, but understanding the phylogenetic algorithms and substitution models, which are based on advanced statistics, is still important for the analysis and interpretation without a guide. Briefly, there are five general steps in carrying out a phylogenetic analysis: (1) sequence data preparation, (2) sequence alignment, (3) choosing a phylogenetic reconstruction method, (4) identification of the best tree, and (5) evaluating the tree. Concepts in this review enable biologists to grasp the basic ideas behind phylogenetic analysis and also help provide a sound basis for discussions with expert phylogeneticists.

Characterization of GM events by insert knowledge adapted re-sequencing approaches

PubMed Central

Yang, Litao; Wang, Congmao; Holst-Jensen, Arne; Morisset, Dany; Lin, Yongjun; Zhang, Dabing

2013-01-01

Detection methods and data from molecular characterization of genetically modified (GM) events are needed by stakeholders of public risk assessors and regulators. Generally, the molecular characteristics of GM events are incomprehensively revealed by current approaches and biased towards detecting transformation vector derived sequences. GM events are classified based on available knowledge of the sequences of vectors and inserts (insert knowledge). Herein we present three insert knowledge-adapted approaches for characterization GM events (TT51-1 and T1c-19 rice as examples) based on paired-end re-sequencing with the advantages of comprehensiveness, accuracy, and automation. The comprehensive molecular characteristics of two rice events were revealed with additional unintended insertions comparing with the results from PCR and Southern blotting. Comprehensive transgene characterization of TT51-1 and T1c-19 is shown to be independent of a priori knowledge of the insert and vector sequences employing the developed approaches. This provides an opportunity to identify and characterize also unknown GM events. PMID:24088728

Characterization of GM events by insert knowledge adapted re-sequencing approaches.

PubMed

Yang, Litao; Wang, Congmao; Holst-Jensen, Arne; Morisset, Dany; Lin, Yongjun; Zhang, Dabing

2013-10-03

Detection methods and data from molecular characterization of genetically modified (GM) events are needed by stakeholders of public risk assessors and regulators. Generally, the molecular characteristics of GM events are incomprehensively revealed by current approaches and biased towards detecting transformation vector derived sequences. GM events are classified based on available knowledge of the sequences of vectors and inserts (insert knowledge). Herein we present three insert knowledge-adapted approaches for characterization GM events (TT51-1 and T1c-19 rice as examples) based on paired-end re-sequencing with the advantages of comprehensiveness, accuracy, and automation. The comprehensive molecular characteristics of two rice events were revealed with additional unintended insertions comparing with the results from PCR and Southern blotting. Comprehensive transgene characterization of TT51-1 and T1c-19 is shown to be independent of a priori knowledge of the insert and vector sequences employing the developed approaches. This provides an opportunity to identify and characterize also unknown GM events.

Multifunctional optical security features based on bacteriorhodopsin

NASA Astrophysics Data System (ADS)

Hampp, Norbert A.; Neebe, Martin; Juchem, Thorsten; Wolperdinger, Markus; Geiger, Markus; Schmuck, Arno

2004-06-01

Bacteriorhodopsin (BR), a photochromic retinal protein, has been developed into a new materials platform for applications in anti-counterfeiting. The combination of three different properties of the material on its molecular level, a light-inducible color change, photochemical data storage and traceability of the protein due to molecular marker sequences make this protein a promising material for security applications. The crystalline structure of the biopigment combines these properties with high stability. As BR is a biological material specialized knowledge for modification, cost- effective production and suitable processing of the material is required. Photochromic BR-based inks have been developed for screen printing, pad printing and ink jet printing. These prints show a high photochromic sensitivity towards variation of illumination. For this reason it is not possible to reproduce the dynamic color by photocopying. In addition to such visual inspection the printed symbols offer the possibility for digital write-once-read-many (WORM) data storage. Photochemical recording is accomplished by a two-photon process. Recording densities in a range from 106 bit/cm2 to 108 bit/cm2 have been achieved. Data structures are stored in a polarization sensitive mode which allows an easy and efficient data encryption.

Frequency Dependent Non- Thermal Effects of Oscillating Electric Fields in the Microwave Region on the Properties of a Solvated Lysozyme System: A Molecular Dynamics Study

PubMed Central

Floros, Stelios; Liakopoulou-Kyriakides, Maria; Karatasos, Kostas

2017-01-01

The use of microwaves in every day’s applications raises issues regarding the non thermal biological effects of microwaves. In this work we employ molecular dynamics simulations to advance further the dielectric studies of protein solutions in the case of lysozyme, taking into consideration possible frequency dependent changes in the structural and dynamic properties of the system upon application of electric field in the microwave region. The obtained dielectric spectra are identical with those derived in our previous work using the Fröhlich-Kirkwood approach in the framework of the linear response theory. Noticeable structural changes in the protein have been observed only at frequencies near its absorption maximum. Concerning Cα position fluctuations, different frequencies affected different regions of the protein sequence. Furthermore, the influence of the field on the kinetics of protein-water as well as on the water-water hydrogen bonds in the first hydration shell has been studied; an extension of the Luzar-Chandler kinetic model was deemed necessary for a better fit of the applied field results and for the estimation of more accurate hydrogen bond lifetime values. PMID:28129348

NMR investigations of molecular dynamics

NASA Astrophysics Data System (ADS)

Palmer, Arthur

2011-03-01

NMR spectroscopy is a powerful experimental approach for characterizing protein conformational dynamics on multiple time scales. The insights obtained from NMR studies are complemented and by molecular dynamics (MD) simulations, which provide full atomistic details of protein dynamics. Homologous mesophilic (E. coli) and thermophilic (T. thermophilus) ribonuclease H (RNase H) enzymes serve to illustrate how changes in protein sequence and structure that affect conformational dynamic processes can be monitored and characterized by joint analysis of NMR spectroscopy and MD simulations. A Gly residue inserted within a putative hinge between helices B and C is conserved among thermophilic RNases H, but absent in mesophilic RNases H. Experimental spin relaxation measurements show that the dynamic properties of T. thermophilus RNase H are recapitulated in E. coli RNase H by insertion of a Gly residue between helices B and C. Additional specific intramolecular interactions that modulate backbone and sidechain dynamical properties of the Gly-rich loop and of the conserved Trp residue flanking the Gly insertion site have been identified using MD simulations and subsequently confirmed by NMR spin relaxation measurements. These results emphasize the importance of hydrogen bonds and local steric interactions in restricting conformational fluctuations, and the absence of such interactions in allowing conformational adaptation to substrate binding.

Molecular Evolution in Historical Perspective.

PubMed

Suárez-Díaz, Edna

2016-12-01

In the 1960s, advances in protein chemistry and molecular genetics provided new means for the study of biological evolution. Amino acid sequencing, nucleic acid hybridization, zone gel electrophoresis, and immunochemistry were some of the experimental techniques that brought about new perspectives to the study of the patterns and mechanisms of evolution. New concepts, such as the molecular evolutionary clock, and the discovery of unexpected molecular phenomena, like the presence of repetitive sequences in eukaryotic genomes, eventually led to the realization that evolution might occur at a different pace at the organismic and the molecular levels, and according to different mechanisms. These developments sparked important debates between defendants of the molecular and organismic approaches. The most vocal confrontations focused on the relation between primates and humans, and the neutral theory of molecular evolution. By the 1980s and 1990s, the construction of large protein and DNA sequences databases, and the development of computer-based statistical tools, facilitated the coming together of molecular and evolutionary biology. Although in its contemporary form the field of molecular evolution can be traced back to the last five decades, the field has deep roots in twentieth century experimental life sciences. For historians of science, the origins and consolidation of molecular evolution provide a privileged field for the study of scientific debates, the relation between technological advances and scientific knowledge, and the connection between science and broader social concerns.

Single Nucleobase Identification Using Biophysical Signatures from Nanoelectronic Quantum Tunneling.

PubMed

Korshoj, Lee E; Afsari, Sepideh; Khan, Sajida; Chatterjee, Anushree; Nagpal, Prashant

2017-03-01

Nanoelectronic DNA sequencing can provide an important alternative to sequencing-by-synthesis by reducing sample preparation time, cost, and complexity as a high-throughput next-generation technique with accurate single-molecule identification. However, sample noise and signature overlap continue to prevent high-resolution and accurate sequencing results. Probing the molecular orbitals of chemically distinct DNA nucleobases offers a path for facile sequence identification, but molecular entropy (from nucleotide conformations) makes such identification difficult when relying only on the energies of lowest-unoccupied and highest-occupied molecular orbitals (LUMO and HOMO). Here, nine biophysical parameters are developed to better characterize molecular orbitals of individual nucleobases, intended for single-molecule DNA sequencing using quantum tunneling of charges. For this analysis, theoretical models for quantum tunneling are combined with transition voltage spectroscopy to obtain measurable parameters unique to the molecule within an electronic junction. Scanning tunneling spectroscopy is then used to measure these nine biophysical parameters for DNA nucleotides, and a modified machine learning algorithm identified nucleobases. The new parameters significantly improve base calling over merely using LUMO and HOMO frontier orbital energies. Furthermore, high accuracies for identifying DNA nucleobases were observed at different pH conditions. These results have significant implications for developing a robust and accurate high-throughput nanoelectronic DNA sequencing technique. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Phenotypic and molecular characterization of Malassezia japonica isolated from psoriasis vulgaris patients.

PubMed

Honnavar, Prasanna; Chakrabarti, Arunaloke; Dogra, Sunil; Handa, Sanjeev; Rudramurthy, Shivaprakash M

2015-03-01

Malassezia species, which are skin colonizers, are being debated as to their pathogenic role in various cutaneous diseases. Species identification of Malassezia is important as particular species have been implicated in or associated with specific diseases. Malassezia japonica, a relatively newly described species, has not been completely characterized owing to the rarity of its isolation. In the present study we describe phenotypic and molecular characterization of six M. japonica strains isolated from patients with psoriasis vulgaris. In contrast to the physiological and biochemical properties of the M. japonica type strain, CBS9348, all our isolates assimilated Tween 20 and showed positive β-glucosidase activity, and the Cremophor EL utilization test was negative. However, the sequences of the D1/D2 region of rDNA, ITS2 and IGS1 regions of all our isolates clustered with the type strain of M. japonica. © 2015 The Authors.

The scope and strength of sex-specific selection in genome evolution

PubMed Central

Wright, A E; Mank, J E

2013-01-01

Males and females share the vast majority of their genomes and yet are often subject to different, even conflicting, selection. Genomic and transcriptomic developments have made it possible to assess sex-specific selection at the molecular level, and it is clear that sex-specific selection shapes the evolutionary properties of several genomic characteristics, including transcription, post-transcriptional regulation, imprinting, genome structure and gene sequence. Sex-specific selection is strongly influenced by mating system, which also causes neutral evolutionary changes that affect different regions of the genome in different ways. Here, we synthesize theoretical and molecular work in order to provide a cohesive view of the role of sex-specific selection and mating system in genome evolution. We also highlight the need for a combined approach, incorporating both genomic data and experimental phenotypic studies, in order to understand precisely how sex-specific selection drives evolutionary change across the genome. PMID:23848139

Molecular Determinants for Antibody Binding on Group 1 House Dust Mite Allergens

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

Chruszcz, Maksymilian; Pomés, Anna; Glesner, Jill

2012-07-11

House dust mites produce potent allergens, Der p 1 and Der f 1, that cause allergic sensitization and asthma. Der p 1 and Der f 1 are cysteine proteases that elicit IgE responses in 80% of mite-allergic subjects and have proinflammatory properties. Their antigenic structure is unknown. Here, we present crystal structures of natural Der p 1 and Der f 1 in complex with a monoclonal antibody, 4C1, which binds to a unique cross-reactive epitope on both allergens associated with IgE recognition. The 4C1 epitope is formed by almost identical amino acid sequences and contact residues. Mutations of the contactmore » residues abrogate mAb 4C1 binding and reduce IgE antibody binding. These surface-exposed residues are molecular targets that can be exploited for development of recombinant allergen vaccines.« less

Magnetic nanoparticles for MR imaging: agents, techniques and cardiovascular applications

PubMed Central

Sosnovik, David E.; Nahrendorf, Matthias; Weissleder, Ralph

2008-01-01

Magnetic nanoparticles (MNP) are playing an increasingly important role in cardiovascular molecular imaging. These agents are superparamagnetic and consist of a central core of iron-oxide surrounded by a carbohydrate or polymer coat. The size, physical properties and pharmacokinetics of MNP make them highly suited to cellular and molecular imaging of atherosclerotic plaque and myocardial injury. MNP have a sensitivity in the nanomolar range and can be detected with T1, T2, T2*, off resonance and steady state free precession sequences. Targeted imaging with MNP is being actively explored and can be achieved through either surface modification or through the attachment of an affinity ligand to the nanoparticle. First generation MNP are already in clinical use and second generation agents, with longer blood half lives, are likely to be approved for routine clinical use in the near future. PMID:18324368

New Methods for Tracking Galaxy and Black Hole Evolution Using Post-Starburst Galaxies

NASA Astrophysics Data System (ADS)

French, Katheryn Decker

2017-08-01

Galaxies in transition from star-forming to quiescence are a natural laboratory for exploring the processes responsible for this evolution. Using a sample of post-starburst galaxies identified to have recently experienced a recent burst of star formation that has now ended, I explore both the fate of the molecular gas that drives star formation and the increased rate of stars disrupted by the central supermassive black hole. Chapter 1 provides an introduction to galaxy evolution through the post-starburst phase and to tidal disruption events, which surprisingly favor post-starburst galaxy hosts. In Chapter 2, I present a survey of the molecular gas properties of 32 post-starburst galaxies traced by CO (1-0) and CO (2-1). In order to accurately put galaxies on an evolutionary sequence, we must select likely progenitors and descendants. We do this by identifying galaxies with similar starburst properties, such as the amount of mass produced in the burst and the burst duration. In Chapter 3, I describe a method to determine the starburst properties and the time elapsed since the starburst ended, and discuss trends in the molecular gas properties of these galaxies with time. In Chapter 4, I present the results of followup observations with ALMA of HCN (1-0) and HCO+ (1-0) in two post-starburst galaxies. CO (1-0) is detected in over half (17/32) the post-starburst sample and the molecular gas mass traced by CO declines on ˜100 Myr timescales after the starburst has ended. HCN (1-0) is not detected in either galaxy targeted, indicating the post-starbursts are now quiescent because of a lack of the denser molecular gas traced by HCN. In Chapter 5 I quantify the increase in TDE rate in quiescent galaxies with strong Balmer absorption to be 30 - 200x higher than in normal galaxies. Using the stellar population fitting method from Chapter 3, I examine possible reasons for the increased TDE rate in post-starburst galaxies in Chapter 6. The TDE rate could be boosted due to a binary supermassive black hole coalescing after a major merger or an increased density of stars or gas remaining near the nucleus after the starburst has ended. In Chapter 7, I present a summary of the findings of this dissertation and an outlook for future work.

Current and future molecular diagnostics for ocular infectious diseases.

PubMed

Doan, Thuy; Pinsky, Benjamin A

2016-11-01

Confirmation of ocular infections can pose great challenges to the clinician. A fundamental limitation is the small amounts of specimen that can be obtained from the eye. Molecular diagnostics can circumvent this limitation and have been shown to be more sensitive than conventional culture. The purpose of this review is to describe new molecular methods and to discuss the applications of next-generation sequencing-based approaches in the diagnosis of ocular infections. Efforts have focused on improving the sensitivity of pathogen detection using molecular methods. This review describes a new molecular target for Toxoplasma gondii-directed polymerase chain reaction assays. Molecular diagnostics for Chlamydia trachomatis and Acanthamoeba species are also discussed. Finally, we describe a hypothesis-free approach, metagenomic deep sequencing, which can detect DNA and RNA pathogens from a single specimen in one test. In some cases, this method can provide the geographic location and timing of the infection. Pathogen-directed PCRs have been powerful tools in the diagnosis of ocular infections for over 20 years. The use of next-generation sequencing-based approaches, when available, will further improve sensitivity of detection with the potential to improve patient care.

Sequence diversity of NanA manifests in distinct enzyme kinetics and inhibitor susceptibility

NASA Astrophysics Data System (ADS)

Xu, Zhongli; von Grafenstein, Susanne; Walther, Elisabeth; Fuchs, Julian E.; Liedl, Klaus R.; Sauerbrei, Andreas; Schmidtke, Michaela

2016-04-01

Streptococcus pneumoniae is the leading pathogen causing bacterial pneumonia and meningitis. Its surface-associated virulence factor neuraminidase A (NanA) promotes the bacterial colonization by removing the terminal sialyl residues from glycoconjugates on eukaryotic cell surface. The predominant role of NanA in the pathogenesis of pneumococci renders it an attractive target for therapeutic intervention. Despite the highly conserved activity of NanA, our alignment of the 11 NanAs revealed the evolutionary diversity of this enzyme. The amino acid substitutions we identified, particularly those in the lectin domain and in the insertion domain next to the catalytic centre triggered our special interest. We synthesised the representative NanAs and the mutagenized derivatives from E. coli for enzyme kinetics study and neuraminidase inhibitor susceptibility test. Via molecular docking we got a deeper insight into the differences between the two major variants of NanA and their influence on the ligand-target interactions. In addition, our molecular dynamics simulations revealed a prominent intrinsic flexibility of the linker between the active site and the insertion domain, which influences the inhibitor binding. Our findings for the first time associated the primary sequence diversity of NanA with the biochemical properties of the enzyme and with the inhibitory efficiency of neuraminidase inhibitors.

Influence of structural variation on nuclear localization of DNA-binding polyamide-fluorophore conjugates.

PubMed

Edelson, Benjamin S; Best, Timothy P; Olenyuk, Bogdan; Nickols, Nicholas G; Doss, Raymond M; Foister, Shane; Heckel, Alexander; Dervan, Peter B

2004-01-01

A pivotal step forward in chemical approaches to controlling gene expression is the development of sequence-specific DNA-binding molecules that can enter live cells and traffic to nuclei unaided. DNA-binding polyamides are a class of programmable, sequence-specific small molecules that have been shown to influence a wide variety of protein-DNA interactions. We have synthesized over 100 polyamide-fluorophore conjugates and assayed their nuclear uptake profiles in 13 mammalian cell lines. The compiled dataset, comprising 1300 entries, establishes a benchmark for the nuclear localization of polyamide-dye conjugates. Compounds in this series were chosen to provide systematic variation in several structural variables, including dye composition and placement, molecular weight, charge, ordering of the aromatic and aliphatic amino-acid building blocks and overall shape. Nuclear uptake does not appear to be correlated with polyamide molecular weight or with the number of imidazole residues, although the positions of imidazole residues affect nuclear access properties significantly. Generally negative determinants for nuclear access include the presence of a beta-Ala-tail residue and the lack of a cationic alkyl amine moiety, whereas the presence of an acetylated 2,4-diaminobutyric acid-turn is a positive factor for nuclear localization. We discuss implications of these data on the design of polyamide-dye conjugates for use in biological systems.

Metallothionein from Wild Populations of the African Catfish Clarias gariepinus: From Sequence, Protein Expression and Metal Binding Properties to Transcriptional Biomarker of Metal Pollution.

PubMed

M'kandawire, Ethel; Mierek-Adamska, Agnieszka; Stürzenbaum, Stephen R; Choongo, Kennedy; Yabe, John; Mwase, Maxwell; Saasa, Ngonda; Blindauer, Claudia A

2017-07-18

Anthropogenic pollution with heavy metals is an on-going concern throughout the world, and methods to monitor release and impact of heavy metals are of high importance. With a view to probe its suitability as molecular biomarker of metal pollution, this study has determined a coding sequence for metallothionein of the African sharptooth catfish Clarias gariepinus . The gene product was recombinantly expressed in Escherichia coli in presence of Zn(II), Cd(II), or Cu, and characterised by Electrospray Ionisation Mass Spectrometry and elemental analysis. C. gariepinus MT displays typical features of fish MTs, including 20 conserved cysteines, and seven bound divalent cations (Zn(II) or Cd(II)) when saturated. Livers from wild C. gariepinus fish collected in all three seasons from four different sites on the Kafue River of Zambia were analysed for their metal contents and for MT expression levels by quantitative PCR. Significant correlations were found between Zn and Cu levels and MT expression in livers, with MT expression clearly highest at the most polluted site, Chililabombwe, which is situated in the Copperbelt region. Based on our findings, hepatic expression of MT from C. gariepinus may be further developed as a major molecular biomarker of heavy metal pollution resulting from mining activities in this region.

Production of antioxidant and ACE-inhibitory peptides from Kluyveromyces marxianus protein hydrolysates: Purification and molecular docking.

PubMed

Mirzaei, Mahta; Mirdamadi, Saeed; Ehsani, Mohamad Reza; Aminlari, Mahmoud

2018-04-01

Kluyveromyces marxianus protein hydrolysates were prepared by two different sonicated-enzymatic (trypsin and chymotrypsin) hydrolysis treatments to obtain antioxidant and ACE-inhibitory peptides. Trypsin and chymotrypsin hydrolysates obtained by 5 h, exhibited the highest antioxidant and ACE-inhibitory activities. After fractionation using ultrafiltration and reverse phase high performance liquid chromatography (RP-HPLC) techniques, two new peptides were identified. One fragment (LL-9, MW = 1180 Da) with the amino acid sequence of Leu-Pro-Glu-Ser-Val-His-Leu-Asp-Lys showed significant ACE inhibitory activity (IC 50  = 22.88 μM) while another peptide fragment (VL-9, MW = 1118 Da) with the amino acid sequence of Val-Leu-Ser-Thr-Ser-Phe-Pro-Pro-Lys showed the highest antioxidant and ACE inhibitory properties (IC 50  = 15.20 μM, 5568 μM TE/mg protein). The molecular docking studies revealed that the ACE inhibitory activities of VL-9 is due to interaction with the S2 (His513, His353, Glu281) and S'1 (Glu162) pockets of ACE and LL-9 can fit perfectly into the S1 (Thr345) and S2 (Tyr520, Lys511, Gln281) pockets of ACE. Copyright © 2017. Published by Elsevier B.V.

Focus on PNA Flexibility and RNA Binding using Molecular Dynamics and Metadynamics

PubMed Central

Verona, Massimiliano Donato; Verdolino, Vincenzo; Palazzesi, Ferruccio; Corradini, Roberto

2017-01-01

Peptide Nucleic Acids (PNAs) can efficiently target DNA or RNA acting as chemical tools for gene regulation. Their backbone modification and functionalization is often used to increase the affinity for a particular sequence improving selectivity. The understanding of the trading forces that lead the single strand PNA to bind the DNA or RNA sequence is preparatory for any further rational design, but a clear and unique description of this process is still not complete. In this paper we report further insights into this subject, by a computational investigation aiming at the characterization of the conformations of a single strand PNA and how these can be correlated to its capability in binding DNA/RNA. Employing Metadynamics we were able to better define conformational pre-organizations of the single strand PNA and γ-modified PNA otherwise unrevealed through classical molecular dynamics. Our simulations driven on backbone modified PNAs lead to the conclusion that this γ-functionalization affects the single strand preorganization and targeting properties to the DNA/RNA, in agreement with circular dichroism (CD) spectra obtained for this class of compounds. MD simulations on PNA:RNA dissociation and association mechanisms allowed to reveal the critical role of central bases and preorganization in the binding process. PMID:28211525

De Novo transcriptome assembly (NGS) of Curcuma longa L. rhizome reveals novel transcripts related to anticancer and antimalarial terpenoids.

PubMed

Annadurai, Ramasamy S; Neethiraj, Ramprasad; Jayakumar, Vasanthan; Damodaran, Anand C; Rao, Sudha Narayana; Katta, Mohan A V S K; Gopinathan, Sreeja; Sarma, Santosh Prasad; Senthilkumar, Vanitha; Niranjan, Vidya; Gopinath, Ashok; Mugasimangalam, Raja C

2013-01-01

Herbal remedies are increasingly being recognised in recent years as alternative medicine for a number of diseases including cancer. Curcuma longa L., commonly known as turmeric is used as a culinary spice in India and in many Asian countries has been attributed to lower incidences of gastrointestinal cancers. Curcumin, a secondary metabolite isolated from the rhizomes of this plant has been shown to have significant anticancer properties, in addition to antimalarial and antioxidant effects. We sequenced the transcriptome of the rhizome of the 3 varieties of Curcuma longa L. using Illumina reversible dye terminator sequencing followed by de novo transcriptome assembly. Multiple databases were used to obtain a comprehensive annotation and the transcripts were functionally classified using GO, KOG and PlantCyc. Special emphasis was given for annotating the secondary metabolite pathways and terpenoid biosynthesis pathways. We report for the first time, the presence of transcripts related to biosynthetic pathways of several anti-cancer compounds like taxol, curcumin, and vinblastine in addition to anti-malarial compounds like artemisinin and acridone alkaloids, emphasizing turmeric's importance as a highly potent phytochemical. Our data not only provides molecular signatures for several terpenoids but also a comprehensive molecular resource for facilitating deeper insights into the transcriptome of C. longa.

De Novo Transcriptome Assembly (NGS) of Curcuma longa L. Rhizome Reveals Novel Transcripts Related to Anticancer and Antimalarial Terpenoids

PubMed Central

Jayakumar, Vasanthan; Damodaran, Anand C.; Rao, Sudha Narayana; Katta, Mohan A. V. S. K.; Gopinathan, Sreeja; Sarma, Santosh Prasad; Senthilkumar, Vanitha; Niranjan, Vidya; Gopinath, Ashok; Mugasimangalam, Raja C.

2013-01-01

Herbal remedies are increasingly being recognised in recent years as alternative medicine for a number of diseases including cancer. Curcuma longa L., commonly known as turmeric is used as a culinary spice in India and in many Asian countries has been attributed to lower incidences of gastrointestinal cancers. Curcumin, a secondary metabolite isolated from the rhizomes of this plant has been shown to have significant anticancer properties, in addition to antimalarial and antioxidant effects. We sequenced the transcriptome of the rhizome of the 3 varieties of Curcuma longa L. using Illumina reversible dye terminator sequencing followed by de novo transcriptome assembly. Multiple databases were used to obtain a comprehensive annotation and the transcripts were functionally classified using GO, KOG and PlantCyc. Special emphasis was given for annotating the secondary metabolite pathways and terpenoid biosynthesis pathways. We report for the first time, the presence of transcripts related to biosynthetic pathways of several anti-cancer compounds like taxol, curcumin, and vinblastine in addition to anti-malarial compounds like artemisinin and acridone alkaloids, emphasizing turmeric's importance as a highly potent phytochemical. Our data not only provides molecular signatures for several terpenoids but also a comprehensive molecular resource for facilitating deeper insights into the transcriptome of C. longa. PMID:23468859

Computational biophysical, biochemical, and evolutionary signature of human R-spondin family proteins, the member of canonical Wnt/β-catenin signaling pathway.

PubMed

Sharma, Ashish Ranjan; Chakraborty, Chiranjib; Lee, Sang-Soo; Sharma, Garima; Yoon, Jeong Kyo; George Priya Doss, C; Song, Dong-Keun; Nam, Ju-Suk

2014-01-01

In human, Wnt/β-catenin signaling pathway plays a significant role in cell growth, cell development, and disease pathogenesis. Four human (Rspo)s are known to activate canonical Wnt/β-catenin signaling pathway. Presently, (Rspo)s serve as therapeutic target for several human diseases. Henceforth, basic understanding about the molecular properties of (Rspo)s is essential. We approached this issue by interpreting the biochemical and biophysical properties along with molecular evolution of (Rspo)s thorough computational algorithm methods. Our analysis shows that signal peptide length is roughly similar in (Rspo)s family along with similarity in aa distribution pattern. In Rspo3, four N-glycosylation sites were noted. All members are hydrophilic in nature and showed alike GRAVY values, approximately. Conversely, Rspo3 contains the maximum positively charged residues while Rspo4 includes the lowest. Four highly aligned blocks were recorded through Gblocks. Phylogenetic analysis shows Rspo4 is being rooted with Rspo2 and similarly Rspo3 and Rspo1 have the common point of origin. Through phylogenomics study, we developed a phylogenetic tree of sixty proteins (n = 60) with the orthologs and paralogs seed sequences. Protein-protein network was also illustrated. Results demonstrated in our study may help the future researchers to unfold significant physiological and therapeutic properties of (Rspo)s in various disease models.

Computational Biophysical, Biochemical, and Evolutionary Signature of Human R-Spondin Family Proteins, the Member of Canonical Wnt/β-Catenin Signaling Pathway

PubMed Central

Sharma, Ashish Ranjan; Lee, Sang-Soo; Yoon, Jeong Kyo; George Priya Doss, C.; Song, Dong-Keun

2014-01-01

In human, Wnt/β-catenin signaling pathway plays a significant role in cell growth, cell development, and disease pathogenesis. Four human (Rspo)s are known to activate canonical Wnt/β-catenin signaling pathway. Presently, (Rspo)s serve as therapeutic target for several human diseases. Henceforth, basic understanding about the molecular properties of (Rspo)s is essential. We approached this issue by interpreting the biochemical and biophysical properties along with molecular evolution of (Rspo)s thorough computational algorithm methods. Our analysis shows that signal peptide length is roughly similar in (Rspo)s family along with similarity in aa distribution pattern. In Rspo3, four N-glycosylation sites were noted. All members are hydrophilic in nature and showed alike GRAVY values, approximately. Conversely, Rspo3 contains the maximum positively charged residues while Rspo4 includes the lowest. Four highly aligned blocks were recorded through Gblocks. Phylogenetic analysis shows Rspo4 is being rooted with Rspo2 and similarly Rspo3 and Rspo1 have the common point of origin. Through phylogenomics study, we developed a phylogenetic tree of sixty proteins (n = 60) with the orthologs and paralogs seed sequences. Protein-protein network was also illustrated. Results demonstrated in our study may help the future researchers to unfold significant physiological and therapeutic properties of (Rspo)s in various disease models. PMID:25276837

Considerations for standardizing predictive molecular pathology for cancer prognosis.

PubMed

Fiorentino, Michelangelo; Scarpelli, Marina; Lopez-Beltran, Antonio; Cheng, Liang; Montironi, Rodolfo

2017-01-01

Molecular tests that were once ancillary to the core business of cyto-histopathology are becoming the most relevant workload in pathology departments after histopathology/cytopathology and before autopsies. This has resulted from innovations in molecular biology techniques, which have developed at an incredibly fast pace. Areas covered: Most of the current widely used techniques in molecular pathology such as FISH, direct sequencing, pyrosequencing, and allele-specific PCR will be replaced by massive parallel sequencing that will not be considered next generation, but rather, will be considered to be current generation sequencing. The pre-analytical steps of molecular techniques such as DNA extraction or sample preparation will be largely automated. Moreover, all the molecular pathology instruments will be part of an integrated workflow that traces the sample from extraction to the analytical steps until the results are reported; these steps will be guided by expert laboratory information systems. In situ hybridization and immunohistochemistry for quantification will be largely digitalized as much as histology will be mostly digitalized rather than viewed using microscopy. Expert commentary: This review summarizes the technical and regulatory issues concerning the standardization of molecular tests in pathology. A vision of the future perspectives of technological changes is also provided.

Quantitative analysis of RNA-protein interactions on a massively parallel array for mapping biophysical and evolutionary landscapes

PubMed Central

Buenrostro, Jason D.; Chircus, Lauren M.; Araya, Carlos L.; Layton, Curtis J.; Chang, Howard Y.; Snyder, Michael P.; Greenleaf, William J.

2015-01-01

RNA-protein interactions drive fundamental biological processes and are targets for molecular engineering, yet quantitative and comprehensive understanding of the sequence determinants of affinity remains limited. Here we repurpose a high-throughput sequencing instrument to quantitatively measure binding and dissociation of MS2 coat protein to >107 RNA targets generated on a flow-cell surface by in situ transcription and inter-molecular tethering of RNA to DNA. We decompose the binding energy contributions from primary and secondary RNA structure, finding that differences in affinity are often driven by sequence-specific changes in association rates. By analyzing the biophysical constraints and modeling mutational paths describing the molecular evolution of MS2 from low- to high-affinity hairpins, we quantify widespread molecular epistasis, and a long-hypothesized structure-dependent preference for G:U base pairs over C:A intermediates in evolutionary trajectories. Our results suggest that quantitative analysis of RNA on a massively parallel array (RNAMaP) relationships across molecular variants. PMID:24727714

Insight into the coordination and the binding sites of Cu(2+) by the histidyl-6-tag using experimental and computational tools.

PubMed

Watly, Joanna; Simonovsky, Eyal; Wieczorek, Robert; Barbosa, Nuno; Miller, Yifat; Kozlowski, Henryk

2014-07-07

His-tags are specific sequences containing six to nine subsequent histydyl residues, and they are used for purification of recombinant proteins by use of IMAC chromatography. Such polyhistydyl tags, often used in molecular biology, can be also found in nature. Proteins containing histidine-rich domains play a critical role in many life functions in both prokaryote and eukaryote organisms. Binding mode and the thermodynamic properties of the system depend on the specific metal ion and the histidine sequence. Despite the wide application of the His-tag for purification of proteins, little is known about the properties of metal-binding to such tag domains. This inspired us to undertake detailed studies on the coordination of Cu(2+) ion to hexa-His-tag. Experiments were performed using the potentiometric, UV-visible, CD, and EPR techniques. In addition, molecular dynamics (MD) simulations and density functional theory (DFT) calculations were applied. The experimental studies have shown that the Cu(2+) ion binds most likely to two imidazoles and one, two, or three amide nitrogens, depending on the pH. The structures and stabilities of the complexes for the Cu(2+)-Ac-(His)6-NH2 system using experimental and computational tools were established. Polymorphic binding states are suggested, with a possibility of the formation of α-helix structure induced by metal ion coordination. Metal ion is bound to various pairs of imidazole moieties derived from the tag with different efficiencies. The coordination sphere around the metal ion is completed by molecules of water. Finally, the Cu(2+) binding by Ac-(His)6-NH2 is much more efficient compared to other multihistidine protein domains.

Molecular epidemiology of echoviruses 11 and 30 in Russia: different properties of genotypes within an enterovirus serotype.

PubMed

Yarmolskaya, Maria S; Shumilina, Elena Yu; Ivanova, Olga E; Drexler, Jan Felix; Lukashev, Alexander N

2015-03-01

Over 100 known enterovirus serotypes differ in their epidemiological and pathogenic properties. Much less is known about variation of these features on a sub-serotype level, such as genotypes. Echovirus 11 (E11) and E30 are amongst the most frequent causative agents of aseptic meningitis. We studied the molecular epidemiology of these pathogens to evaluate potential epidemiological and pathogenic dissimilarities of their genotypes. The complete VP1 genome region was sequenced for 97 E11 and 62 E30 isolates collected in Russia from 2008 to 2012, and they were studied in comparison with all 140 E11 and 432 E30 sequences available in GenBank. A geographic pattern of genotype prevalence was observed for both types. Russian E11 isolates belonged mainly to A genotype, which is common in Asia, and D5, which is predominant in Europe. For E30, genotype III by classification of Ke et al. (2011), also termed genotype a by Bailly et al. (2009), was endemic in Russia from 2003 to 2012, while it was not detected in Europe and North America during this time. The E30 genotypes VI-B, VI-G, and VI-H (e, f and h) were regularly introduced from different countries, became predominant and vanished after no more than 4years. In addition to geographic patterns, E11 genotypes also differed by isolation source. Genotype A2 viruses were significantly more often found in sewage, compared to genotype D5 that was isolated from both sewage and human samples. In addition, there was evidence of a different capacity for international transfers among E11 GtA subclusters. Copyright © 2015 Elsevier B.V. All rights reserved.

[Complete genome sequencing and analyses of rabies viruses isolated from wild animals (Chinese Ferret-Badger) in Zhejiang province].

PubMed

Lei, Yong-Liang; Wang, Xiao-Guang; Liu, Fu-Ming; Chen, Xiu-Ying; Ye, Bi-Feng; Mei, Jian-Hua; Lan, Jin-Quan; Tang, Qing

2009-08-01

Based on sequencing the full-length genomes of two Chinese Ferret-Badger, we analyzed the properties of rabies viruses genetic variation in molecular level to get information on prevalence and variation of rabies viruses in Zhejiang, and to enrich the genome database of rabies viruses street strains isolated from Chinese wildlife. Overlapped fragments were amplified by RT-PCR and full-length genomes were assembled to analyze the nucleotide and deduced protein similarities and phylogenetic analyses of the N genes from Chinese Ferret-Badger, sika deer, vole, dog. Vaccine strains were then determined. The two full-length genomes were completely sequenced to find out that they had the same genetic structure with 11 923 nts including 58 nts-Leader, 1353 nts-NP, 894 nts-PP, 609 nts-MP, 1575 nts-GP, 6386 nts-LP, and 2, 5, 5 nts- intergenic regions (IGRs), 423 nts-Pseudogene-like sequence (Psi), 70 nts-Trailer. The two full-length genomes were in accordance with the properties of Rhabdoviridae Lyssa virus by blast and multi-sequence alignment. The nucleotide and amino acid sequences among Chinese strains had the highest similarity, especially among animals of the same species. Of the two full-length genomes, the similarity in amino acid level was dramatically higher than that in nucleotide level, so that the nucleotide mutations happened in these two genomes were most probably as synonymous mutations. Compared to the referenced rabies viruses, the lengths of the five protein coding regions did not show any changes or recombination, but only with a few-point mutations. It was evident that the five proteins appeared to be stable. The variation sites and types of the two ferret badgers genomes were similar to the referenced vaccine or street strains. The two strains were genotype 1 according to the multi-sequence and phylogenetic analyses, which possessing the distinct geographyphic characteristics of China. All the evidence suggested a cue that these two ferret badgers rabies viruses were likely to be street virus that already circulating in wildlife.

Chemical characterization of the early evolutionary phases of high-mass star-forming regions

NASA Astrophysics Data System (ADS)

Gerner, Thomas

2014-10-01

The formation of high-mass stars is a very complex process and up to date no comprehensive theory about it exists. This thesis studies the early stages of high-mass star-forming regions and employs astrochemistry as a tool to probe their different physical conditions. We split the evolutionary sequence into four observationally motivated stages that are based on a classification proposed in the literature. The sequence is characterized by an increase of the temperatures and densities that strongly influences the chemistry in the different stages. We observed a sample of 59 high-mass star-forming regions that cover the whole sequence and statistically characterized the chemical compositions of the different stages. We determined average column densities of 18 different molecular species and found generally increasing abundances with stage. We fitted them for each stage with a 1D model, such that the result of the best fit to the previous stage was used as new input for the following. This is a unique approach and allowed us to infer physical properties like the temperature and density structure and yielded a typical chemical lifetime for the high-mass star-formation process of 1e5 years. The 18 analyzed molecular species also included four deuterated molecules whose chemistry is particularly sensitive to thermal history and thus is a promising tool to infer chemical ages. We found decreasing trends of the D/H ratios with evolutionary stage for 3 of the 4 molecular species and that the D/H ratio depends more on the fraction of warm and cold gas than on the total amount of gas. That indicates different chemical pathways for the different molecules and confirms the potential use of deuterated species as chemical age indicators. In addition, we mapped a low-mass star forming region in order to study the cosmic ray ionization rate, which is an important parameter in chemical models. While in chemical models it is commonly fixed, we found that it ! strongly varies with environment.

Genetic characterization of the hemagglutinin genes of wild-type measles virus circulating in china, 1993-2009.

PubMed

Xu, Songtao; Zhang, Yan; Zhu, Zhen; Liu, Chunyu; Mao, Naiying; Ji, Yixin; Wang, Huiling; Jiang, Xiaohong; Li, Chongshan; Tang, Wei; Feng, Daxing; Wang, Changyin; Zheng, Lei; Lei, Yue; Ling, Hua; Zhao, Chunfang; Ma, Yan; He, Jilan; Wang, Yan; Li, Ping; Guan, Ronghui; Zhou, Shujie; Zhou, Jianhui; Wang, Shuang; Zhang, Hong; Zheng, Huanying; Liu, Leng; Ma, Hemuti; Guan, Jing; Lu, Peishan; Feng, Yan; Zhang, Yanjun; Zhou, Shunde; Xiong, Ying; Ba, Zhuoma; Chen, Hui; Yang, Xiuhui; Bo, Fang; Ma, Yujie; Liang, Yong; Lei, Yake; Gu, Suyi; Liu, Wei; Chen, Meng; Featherstone, David; Jee, Youngmee; Bellini, William J; Rota, Paul A; Xu, Wenbo

2013-01-01

China experienced several large measles outbreaks in the past two decades, and a series of enhanced control measures were implemented to achieve the goal of measles elimination. Molecular epidemiologic surveillance of wild-type measles viruses (MeV) provides valuable information about the viral transmission patterns. Since 1993, virologic surveillnace has confirmed that a single endemic genotype H1 viruses have been predominantly circulating in China. A component of molecular surveillance is to monitor the genetic characteristics of the hemagglutinin (H) gene of MeV, the major target for virus neutralizing antibodies. Analysis of the sequences of the complete H gene from 56 representative wild-type MeV strains circulating in China during 1993-2009 showed that the H gene sequences were clustered into 2 groups, cluster 1 and cluster 2. Cluster1 strains were the most frequently detected cluster and had a widespread distribution in China after 2000. The predicted amino acid sequences of the H protein were relatively conserved at most of the functionally significant amino acid positions. However, most of the genotype H1 cluster1 viruses had an amino acid substitution (Ser240Asn), which removed a predicted N-linked glycosylation site. In addition, the substitution of Pro397Leu in the hemagglutinin noose epitope (HNE) was identified in 23 of 56 strains. The evolutionary rate of the H gene of the genotype H1 viruses was estimated to be approximately 0.76×10(-3) substitutions per site per year, and the ratio of dN to dS (dN/dS) was <1 indicating the absence of selective pressure. Although H genes of the genotype H1 strains were conserved and not subjected to selective pressure, several amino acid substitutions were observed in functionally important positions. Therefore the antigenic and genetic properties of H genes of wild-type MeVs should be monitored as part of routine molecular surveillance for measles in China.

Testing the molecular clock using mechanistic models of fossil preservation and molecular evolution.

PubMed

Warnock, Rachel C M; Yang, Ziheng; Donoghue, Philip C J

2017-06-28

Molecular sequence data provide information about relative times only, and fossil-based age constraints are the ultimate source of information about absolute times in molecular clock dating analyses. Thus, fossil calibrations are critical to molecular clock dating, but competing methods are difficult to evaluate empirically because the true evolutionary time scale is never known. Here, we combine mechanistic models of fossil preservation and sequence evolution in simulations to evaluate different approaches to constructing fossil calibrations and their impact on Bayesian molecular clock dating, and the relative impact of fossil versus molecular sampling. We show that divergence time estimation is impacted by the model of fossil preservation, sampling intensity and tree shape. The addition of sequence data may improve molecular clock estimates, but accuracy and precision is dominated by the quality of the fossil calibrations. Posterior means and medians are poor representatives of true divergence times; posterior intervals provide a much more accurate estimate of divergence times, though they may be wide and often do not have high coverage probability. Our results highlight the importance of increased fossil sampling and improved statistical approaches to generating calibrations, which should incorporate the non-uniform nature of ecological and temporal fossil species distributions. © 2017 The Authors.

Molecular characterization and phylogenetic analysis of Citrus viroid VI variants from citrus in China

USDA-ARS?s Scientific Manuscript database

Citrus viroid VI (CVd-VI) was originally found from citrus and persimmon in Japan. We report here the identification and molecular characterization of CVd-VI from four production regions of China. A total of 90 cDNA clones from nine infected citrus cultivars were sequenced. The sequence homologies o...

Giardia lamblia: Molecular Studies of an Early Branching Eukaryote

USDA-ARS?s Scientific Manuscript database

The rapid advance in our understanding of the biology of Giardia lamblia over the last several years is due in part to the complete DNA sequencing of the 11.7 Mb genome of this diplomonad. Insight on the molecular nature of G. lamblia has been gained by searching the genome using query sequences fr...

Molecular approaches to Taenia asiatica.

PubMed

Jeon, Hyeong-Kyu; Eom, Keeseon S

2013-02-01

Taenia solium, T. saginata, and T. asiatica are taeniid tapeworms that cause taeniasis in humans and cysticercosis in intermediate host animals. Taeniases remain an important public health concerns in the world. Molecular diagnostic methods using PCR assays have been developed for rapid and accurate detection of human infecting taeniid tapeworms, including the use of sequence-specific DNA probes, PCR-RFLP, and multiplex PCR. More recently, DNA diagnosis using PCR based on histopathological specimens such as 10% formalin-fixed paraffin-embedded and stained sections mounted on slides has been applied to cestode infections. The mitochondrial gene sequence is believed to be a very useful molecular marker for not only studying evolutionary relationships among distantly related taxa, but also for investigating the phylo-biogeography of closely related species. The complete sequence of the human Taenia tapeworms mitochondrial genomes were determined, and its organization and structure were compared to other human-tropic Taenia tapeworms for which complete mitochondrial sequence data were available. The multiplex PCR assay with the Ta4978F, Ts5058F, Tso7421F, and Rev7915 primers will be useful for differential diagnosis, molecular characterization, and epidemiological surveys of human Taenia tapeworms.

Amyloid-like self-assembly of peptide sequences from the adenovirus fiber shaft: insights from molecular dynamics simulations.

PubMed

Tamamis, Phanourios; Kasotakis, Emmanouil; Mitraki, Anna; Archontis, Georgios

2009-11-26

The self-assembly of peptides and proteins into nanostructures is related to the fundamental problems of protein folding and misfolding and has potential applications in medicine, materials science and nanotechnology. Natural peptides, corresponding to sequence repeats from self-assembling proteins, may constitute elementary building blocks of such nanostructures. In this work, we study by implicit-solvent replica-exchange simulations the self-assembly of two amyloidogenic sequences derived from the naturally occurring fiber shaft of the adenovirus, the octapeptide NSGAITIG (asparagine-serine-glycine-alanine-isoleucine-threonine-isoleucine-glycine) and its hexapeptide counterpart, GAITIG. In accordance with their amyloidogenic capacity, both peptides form readily intermolecular beta-sheets, stabilized by extensive main- and side-chain contacts involving the C-terminal moieties (segments 3-8 and 2-6, respectively). The structural and energetic properties of these sheets are analyzed extensively. The N-terminal residues Asn1 and Ser2 of the octapeptide remain disordered in the sheets, suggesting that these residues are exposed at the exterior of the fibrils and accessible. On the basis of insight provided by the simulations, cysteine residues were recently substituted at positions 1 and 2 of NSGAITIG; the newly designed peptides maintain their amyloidogenic properties and can bind to silver, gold and platinum nanoparticles [Kasotakis et al. Biopolymers 2009, 92, 164-172]. Computational investigation can identify suitable positions for rational modification of peptide building blocks, aiming at the fabrication of novel biomaterials.

The influence of specific binding of collagen-silk chimeras to silk biomaterials on hMSC behavior

PubMed Central

An, Bo; DesRochers, Teresa M.; Qin, Guokui; Xia, Xiaoxia; Thiagarajan, Geetha; Brodsky, Barbara; Kaplan, David

2012-01-01

Collagen-like proteins in the bacteria Streptococcus pyogenes adopt a triple-helix structure with a thermal stability similar to that of animal collagens, can be expressed in high yield in E. coli and can be easily modified through molecular biology techniques. However, potential applications for such recombinant collagens are limited by their lack of higher order structure to achieve the physical properties needed for most biomaterials. To overcome this problem, the S. pyrogenes collagen domain was fused to a repetitive Bombyx mori silk consensus sequence, as a strategy to direct specific non-covalent binding onto solid silk materials whose superior stability, mechanical and material properties have been previously established. This approach resulted in the successful binding of these new collagen-silk chimeric proteins to silk films and porous scaffolds, and the binding affinity could be controlled by varying the number of repeats in the silk sequence. To explore the potential of collagen-silk chimera for regulating biological activity, integrin (Int) and fibronectin (Fn) binding sequences from mammalian collagens were introduced into the bacterial collagen domain. The attachment of bioactive collagen-silk chimeras to solid silk biomaterials promoted hMSC spreading and proliferation substantially in comparison to the controls. The ability to combine the biomaterial features of silk with the biological activities of collagen allowed more rapid cell interactions with silk-based biomaterials, improved regulation of stem cell growth and differentiation, as well as the formation of artificial extracellular matrices useful for tissue engineering applications. PMID:23088839

Computational analysis of protein-protein interfaces involving an alpha helix: insights for terphenyl-like molecules binding.

PubMed

Isvoran, Adriana; Craciun, Dana; Martiny, Virginie; Sperandio, Olivier; Miteva, Maria A

2013-06-14

Protein-Protein Interactions (PPIs) are key for many cellular processes. The characterization of PPI interfaces and the prediction of putative ligand binding sites and hot spot residues are essential to design efficient small-molecule modulators of PPI. Terphenyl and its derivatives are small organic molecules known to mimic one face of protein-binding alpha-helical peptides. In this work we focus on several PPIs mediated by alpha-helical peptides. We performed computational sequence- and structure-based analyses in order to evaluate several key physicochemical and surface properties of proteins known to interact with alpha-helical peptides and/or terphenyl and its derivatives. Sequence-based analysis revealed low sequence identity between some of the analyzed proteins binding alpha-helical peptides. Structure-based analysis was performed to calculate the volume, the fractal dimension roughness and the hydrophobicity of the binding regions. Besides the overall hydrophobic character of the binding pockets, some specificities were detected. We showed that the hydrophobicity is not uniformly distributed in different alpha-helix binding pockets that can help to identify key hydrophobic hot spots. The presence of hydrophobic cavities at the protein surface with a more complex shape than the entire protein surface seems to be an important property related to the ability of proteins to bind alpha-helical peptides and low molecular weight mimetics. Characterization of similarities and specificities of PPI binding sites can be helpful for further development of small molecules targeting alpha-helix binding proteins.

The impact of age, biogenesis, and genomic clustering on Drosophila microRNA evolution

PubMed Central

Mohammed, Jaaved; Flynt, Alex S.; Siepel, Adam; Lai, Eric C.

2013-01-01

The molecular evolutionary signatures of miRNAs inform our understanding of their emergence, biogenesis, and function. The known signatures of miRNA evolution have derived mostly from the analysis of deeply conserved, canonical loci. In this study, we examine the impact of age, biogenesis pathway, and genomic arrangement on the evolutionary properties of Drosophila miRNAs. Crucial to the accuracy of our results was our curation of high-quality miRNA alignments, which included nearly 150 corrections to ortholog calls and nucleotide sequences of the global 12-way Drosophilid alignments currently available. Using these data, we studied primary sequence conservation, normalized free-energy values, and types of structure-preserving substitutions. We expand upon common miRNA evolutionary patterns that reflect fundamental features of miRNAs that are under functional selection. We observe that melanogaster-subgroup-specific miRNAs, although recently emerged and rapidly evolving, nonetheless exhibit evolutionary signatures that are similar to well-conserved miRNAs and distinct from other structured noncoding RNAs and bulk conserved non-miRNA hairpins. This provides evidence that even young miRNAs may be selected for regulatory activities. More strikingly, we observe that mirtrons and clustered miRNAs both exhibit distinct evolutionary properties relative to solo, well-conserved miRNAs, even after controlling for sequence depth. These studies highlight the previously unappreciated impact of biogenesis strategy and genomic location on the evolutionary dynamics of miRNAs, and affirm that miRNAs do not evolve as a unitary class. PMID:23882112

Evolutionary and Functional Relationships in the Truncated Hemoglobin Family.

PubMed

Bustamante, Juan P; Radusky, Leandro; Boechi, Leonardo; Estrin, Darío A; Ten Have, Arjen; Martí, Marcelo A

2016-01-01

Predicting function from sequence is an important goal in current biological research, and although, broad functional assignment is possible when a protein is assigned to a family, predicting functional specificity with accuracy is not straightforward. If function is provided by key structural properties and the relevant properties can be computed using the sequence as the starting point, it should in principle be possible to predict function in detail. The truncated hemoglobin family presents an interesting benchmark study due to their ubiquity, sequence diversity in the context of a conserved fold and the number of characterized members. Their functions are tightly related to O2 affinity and reactivity, as determined by the association and dissociation rate constants, both of which can be predicted and analyzed using in-silico based tools. In the present work we have applied a strategy, which combines homology modeling with molecular based energy calculations, to predict and analyze function of all known truncated hemoglobins in an evolutionary context. Our results show that truncated hemoglobins present conserved family features, but that its structure is flexible enough to allow the switch from high to low affinity in a few evolutionary steps. Most proteins display moderate to high oxygen affinities and multiple ligand migration paths, which, besides some minor trends, show heterogeneous distributions throughout the phylogenetic tree, again suggesting fast functional adaptation. Our data not only deepens our comprehension of the structural basis governing ligand affinity, but they also highlight some interesting functional evolutionary trends.

Evolutionary and Functional Relationships in the Truncated Hemoglobin Family

PubMed Central

Bustamante, Juan P.; Radusky, Leandro; Boechi, Leonardo; Estrin, Darío A.; ten Have, Arjen; Martí, Marcelo A.

2016-01-01

Predicting function from sequence is an important goal in current biological research, and although, broad functional assignment is possible when a protein is assigned to a family, predicting functional specificity with accuracy is not straightforward. If function is provided by key structural properties and the relevant properties can be computed using the sequence as the starting point, it should in principle be possible to predict function in detail. The truncated hemoglobin family presents an interesting benchmark study due to their ubiquity, sequence diversity in the context of a conserved fold and the number of characterized members. Their functions are tightly related to O2 affinity and reactivity, as determined by the association and dissociation rate constants, both of which can be predicted and analyzed using in-silico based tools. In the present work we have applied a strategy, which combines homology modeling with molecular based energy calculations, to predict and analyze function of all known truncated hemoglobins in an evolutionary context. Our results show that truncated hemoglobins present conserved family features, but that its structure is flexible enough to allow the switch from high to low affinity in a few evolutionary steps. Most proteins display moderate to high oxygen affinities and multiple ligand migration paths, which, besides some minor trends, show heterogeneous distributions throughout the phylogenetic tree, again suggesting fast functional adaptation. Our data not only deepens our comprehension of the structural basis governing ligand affinity, but they also highlight some interesting functional evolutionary trends. PMID:26788940

Identifying the pattern of molecular evolution for Zaire ebolavirus in the 2014 outbreak in West Africa.

PubMed

Liu, Si-Qing; Deng, Cheng-Lin; Yuan, Zhi-Ming; Rayner, Simon; Zhang, Bo

2015-06-01

The current Ebola virus disease (EVD) epidemic has killed more than all previous Ebola outbreaks combined and, even as efforts appear to be bringing the outbreak under control, the threat of reemergence remains. The availability of new whole-genome sequences from West Africa in 2014 outbreak, together with those from the earlier outbreaks, provide an opportunity to investigate the genetic characteristics, the epidemiological dynamics and the evolutionary history for Zaire ebolavirus (ZEBOV). To investigate the evolutionary properties of ZEBOV in this outbreak, we examined amino acid mutations, positive selection, and evolutionary rates on the basis of 123 ZEBOV genome sequences. The estimated phylogenetic relationships within ZEBOV revealed that viral sequences from the same period or location formed a distinct cluster. The West Africa viruses probably derived from Middle Africa, consistent with results from previous studies. Analysis of the seven protein regions of ZEBOV revealed evidence of positive selection acting on the GP and L genes. Interestingly, all putatively positive-selected sites identified in the GP are located within the mucin-like domain of the solved structure of the protein, suggesting a possible role in the immune evasion properties of ZEBOV. Compared with earlier outbreaks, the evolutionary rate of GP gene was estimated to significantly accelerate in the 2014 outbreak, suggesting that more ZEBOV variants are generated for human to human transmission during this sweeping epidemic. However, a more balanced sample set and next generation sequencing datasets would help achieve a clearer understanding at the genetic level of how the virus is evolving and adapting to new conditions. Copyright © 2015 Elsevier B.V. All rights reserved.

Molecular determinants of enzyme cold adaptation: comparative structural and computational studies of cold- and warm-adapted enzymes.

PubMed

Papaleo, Elena; Tiberti, Matteo; Invernizzi, Gaetano; Pasi, Marco; Ranzani, Valeria

2011-11-01

The identification of molecular mechanisms underlying enzyme cold adaptation is a hot-topic both for fundamental research and industrial applications. In the present contribution, we review the last decades of structural computational investigations on cold-adapted enzymes in comparison to their warm-adapted counterparts. Comparative sequence and structural studies allow the definition of a multitude of adaptation strategies. Different enzymes carried out diverse mechanisms to adapt to low temperatures, so that a general theory for enzyme cold adaptation cannot be formulated. However, some common features can be traced in dynamic and flexibility properties of these enzymes, as well as in their intra- and inter-molecular interaction networks. Interestingly, the current data suggest that a family-centered point of view is necessary in the comparative analyses of cold- and warm-adapted enzymes. In fact, enzymes belonging to the same family or superfamily, thus sharing at least the three-dimensional fold and common features of the functional sites, have evolved similar structural and dynamic patterns to overcome the detrimental effects of low temperatures.

Studying the influence of stem composition in pH-sensitive molecular beacons onto their sensing properties.

PubMed

Dembska, Anna; Kierzek, Elzbieta; Juskowiak, Bernard

2017-10-16

Intracellular sensing using fluorescent molecular beacons is a potentially useful strategy for real-time, in vivo monitoring of important cellular events. This work is focused on evaluation of pyrene excimer signaling molecular beacons (MBs) for the monitoring of pH changes in vitro as well as inside living cells. The recognition element in our MB called pHSO (pH-sensitive oligonucleotide) is the loop enclosing cytosine-rich fragment that is able to form i-motif structure in a specific pH range. However, alteration of a sequence of the 6 base pairs containing stem of MB allowed the design of pHSO probes that exhibited different dynamic pH range and possessed slightly different transition midpoint between i-motif and open loop configuration. Moreover, this conformational transition was accompanied by spectral changes showing developed probes different pyrene excimer-monomer emission ratio triggered by pH changes. The potential of these MBs for intracellular pH sensing is demonstrated on the example of HeLa cells line. Copyright © 2017 Elsevier B.V. All rights reserved.

Iterative divergent/convergent doubling approach to linear conjugated oligomers. A rapid route to a 128 A long potential molecular wire

NASA Astrophysics Data System (ADS)

Tour, James M.; Schumm, Jeffrey S.; Pearson, Darren L.

1994-06-01

Described is the synthesis of oligo (2-ethylphenylene ethynylene)s and oligo (2-(3'ethylheptyl) phenylene ethynylene)s via an iterative divergent convergent approach. Synthesized were the monomer, dimer, tetramer, and octamer of the ethyl derivative and the monomer, dimer, tetramer, octamer, and 16-mer of the ethylheptyl derivative. The 16-mer is 128 A long. At each stage in the iteration, the length of the framework doubles. Only three sets of reaction conditions are needed for the entire iterative synthetic sequence; an iodination, a protodesilylation, and a Pd/Cu-catalyzed cross coupling. The oligomers were characterized spectroscopically and by mass spectrometry. The optical properties are presented which show the stage of optical absorbance saturation. The size exclusion chromatography values for the number average weights, relative to polystyrene, illustrate the tremendous differences in the hydrodynamic volume of these rigid rod oligomers verses the random coils of polystyrene. These differences become quite apparent at the octamer stage. These oligomers may act as molecular wires in molecular electronic devices and they also serve as useful models for understanding related bulk polymers.

Inducing β-Sheets Formation in Synthetic Spider Silk Fibers by Aqueous Post-Spin Stretching

PubMed Central

Hinman, Michael B.; Holland, Gregory P.; Yarger, Jeffery L.; Lewis, Randolph V.

2012-01-01

As a promising biomaterial with numerous potential applications, various types of synthetic spider silk fibers have been produced and studied in an effort to produce manmade fibers with mechanical and physical properties comparable to those of native spider silk. In this study, two recombinant proteins based on Nephila clavipes Major ampullate Spidroin 1 (MaSp1) consensus repeat sequence were expressed and spun into fibers. Mechanical test results showed that fiber spun from the higher molecular weight protein had better overall mechanical properties (70 KD versus 46 KD), whereas postspin stretch treatment in water helped increase fiber tensile strength significantly. Carbon-13 solid-state NMR studies of those fibers further revealed that the postspin stretch in water promoted protein molecule rearrangement and the formation of β-sheets in the polyalanine region of the silk. The rearrangement correlated with improved fiber mechanical properties and indicated that postspin stretch is key to helping the spider silk proteins in the fiber form correct secondary structures, leading to better quality fibers. PMID:21574576

What is biodiversity? Stepping forward from barcoding to understanding biological differences.

PubMed

Nikinmaa, Mikko

2014-10-01

This opinion paper gives personal views of the direction that cataloguing biodiversity should be going in. Although molecular taxonomy enables rapid and high throughput identification of species, it needs to be anchored to traditional taxonomy, because without information of actual biological properties of species, DNA barcoding just reports differences in selected DNA sequences, which need not have anything to do with the biological properties of the organisms, and the reasons for the development of the species. Since functional differences are the most common reason behind species differences, the future of cataloguing biodiversity and biodiversity research is, in my opinion, in trying to integrate genomic research to comparative physiology in order to be able to evaluate which functional properties have likely been important in generating biodiversity. This task is overwhelming, and requires forgetting the traditional disciplines. Further, major problems associated with the present-day treatment of genomic data are presented from my viewpoint. Copyright © 2014 Elsevier B.V. All rights reserved.

Viscoelastic properties of dendrimers in the melt from nonequlibrium molecular dynamics

NASA Astrophysics Data System (ADS)

Bosko, Jaroslaw T.; Todd, B. D.; Sadus, Richard J.

2004-12-01

The viscoelastic properties of dendrimers of generation 1-4 are studied using nonequilibrium molecular dynamics. Flow properties of dendrimer melts under shear are compared to systems composed of linear chain polymers of the same molecular weight, and the influence of molecular architecture is discussed. Rheological material properties, such as the shear viscosity and normal stress coefficients, are calculated and compared for both systems. We also calculate and compare the microscopic properties of both linear chain and dendrimer molecules, such as their molecular alignment, order parameters and rotational velocities. We find that the highly symmetric shape of dendrimers and their highly constrained geometry allows for substantial differences in their material properties compared to traditional linear polymers of equivalent molecular weight.

Recent Advancement of the Molecular Diagnosis in Pediatric Brain Tumor.

PubMed

Bae, Jeong-Mo; Won, Jae-Kyung; Park, Sung-Hye

2018-05-01

Recent discoveries of brain tumor-related genes and fast advances in genomic testing technologies have led to the era of molecular diagnosis of brain tumor. Molecular profiling of brain tumor became the significant step in the diagnosis, the prediction of prognosis and the treatment of brain tumor. Because traditional molecular testing methods have limitations in time and cost for multiple gene tests, next-generation sequencing technologies are rapidly introduced into clinical practice. Targeted sequencing panels using these technologies have been developed for brain tumors. In this article, focused on pediatric brain tumor, key discoveries of brain tumor-related genes are reviewed and cancer panels used in the molecular profiling of brain tumor are discussed.

Recent Advancement of the Molecular Diagnosis in Pediatric Brain Tumor

PubMed Central

Bae, Jeong-Mo; Won, Jae-Kyung; Park, Sung-Hye

2018-01-01

Recent discoveries of brain tumor-related genes and fast advances in genomic testing technologies have led to the era of molecular diagnosis of brain tumor. Molecular profiling of brain tumor became the significant step in the diagnosis, the prediction of prognosis and the treatment of brain tumor. Because traditional molecular testing methods have limitations in time and cost for multiple gene tests, next-generation sequencing technologies are rapidly introduced into clinical practice. Targeted sequencing panels using these technologies have been developed for brain tumors. In this article, focused on pediatric brain tumor, key discoveries of brain tumor-related genes are reviewed and cancer panels used in the molecular profiling of brain tumor are discussed. PMID:29742887

Generation of Synthetic Copolymer Libraries by Combinatorial Assembly on Nucleic Acid Templates.

PubMed

Kong, Dehui; Yeung, Wayland; Hili, Ryan

2016-07-11

Recent advances in nucleic acid-templated copolymerization have expanded the scope of sequence-controlled synthetic copolymers beyond the molecular architectures witnessed in nature. This has enabled the power of molecular evolution to be applied to synthetic copolymer libraries to evolve molecular function ranging from molecular recognition to catalysis. This Review seeks to summarize different approaches available to generate sequence-defined monodispersed synthetic copolymer libraries using nucleic acid-templated polymerization. Key concepts and principles governing nucleic acid-templated polymerization, as well as the fidelity of various copolymerization technologies, will be described. The Review will focus on methods that enable the combinatorial generation of copolymer libraries and their molecular evolution for desired function.

Ligand-mediated protein degradation reveals functional conservation among sequence variants of the CUL4-type E3 ligase substrate receptor cereblon.

PubMed

Akuffo, Afua A; Alontaga, Aileen Y; Metcalf, Rainer; Beatty, Matthew S; Becker, Andreas; McDaniel, Jessica M; Hesterberg, Rebecca S; Goodheart, William E; Gunawan, Steven; Ayaz, Muhammad; Yang, Yan; Karim, Md Rezaul; Orobello, Morgan E; Daniel, Kenyon; Guida, Wayne; Yoder, Jeffrey A; Rajadhyaksha, Anjali M; Schönbrunn, Ernst; Lawrence, Harshani R; Lawrence, Nicholas J; Epling-Burnette, Pearlie K

2018-04-20

Upon binding to thalidomide and other immunomodulatory drugs, the E3 ligase substrate receptor cereblon (CRBN) promotes proteosomal destruction by engaging the DDB1-CUL4A-Roc1-RBX1 E3 ubiquitin ligase in human cells but not in mouse cells, suggesting that sequence variations in CRBN may cause its inactivation. Therapeutically, CRBN engagers have the potential for broad applications in cancer and immune therapy by specifically reducing protein expression through targeted ubiquitin-mediated degradation. To examine the effects of defined sequence changes on CRBN's activity, we performed a comprehensive study using complementary theoretical, biophysical, and biological assays aimed at understanding CRBN's nonprimate sequence variations. With a series of recombinant thalidomide-binding domain (TBD) proteins, we show that CRBN sequence variants retain their drug-binding properties to both classical immunomodulatory drugs and dBET1, a chemical compound and targeting ligand designed to degrade bromodomain-containing 4 (BRD4) via a CRBN-dependent mechanism. We further show that dBET1 stimulates CRBN's E3 ubiquitin-conjugating function and degrades BRD4 in both mouse and human cells. This insight paves the way for studies of CRBN-dependent proteasome-targeting molecules in nonprimate models and provides a new understanding of CRBN's substrate-recruiting function. © 2018 by The American Society for Biochemistry and Molecular Biology, Inc.

Unusually weak oxygen binding, physical properties, partial sequence, autoxidation rate and a potential phosphorylation site of beluga whale (Delphinapterus leucas) myoglobin.

PubMed

Stewart, J M; Blakely, J A; Karpowicz, P A; Kalanxhi, E; Thatcher, B J; Martin, B M

2004-03-01

We purified myoglobin from beluga whale (Delphinapterus leucas) muscle (longissimus dorsi) with size exclusion and cation exchange chromatographies. The molecular mass was determined by mass spectrometry (17,081 Da) and the isoelectric pH (9.4) by capillary isoelectric focusing. The near-complete amino acid sequence was determined and a phylogeny indicated that beluga was in the same clad as Dall's and harbor porpoises. There were consensus motifs for a phosphorylation site on the protein surface with the most likely site at serine-117. This motif was common to all cetacean myoglobins examined. Two oxygen-binding studies at 37 degrees C indicated dissociation constants (20.5 and 23.6 microM) 5.7-6.6 times larger than horse myoglobin (3.6 microM). The autoxidation rate of beluga myoglobin at 37 degrees C, pH 7.2 was 0.218+/-0.028 h(-1), 1/3 larger than reported for myoglobin of terrestrial mammals. There was no clear sequence change to explain the difference in oxygen binding or autoxidation although substitutions (N66 and T67) in an invariant rich sequence (HGNTV) distal to the heme may play a role. Structural models based on the protein sequence and constructed on topologies of known templates (horse and sperm whale crystal structures) were not adequate to assess perturbation of the heme pocket.

Exome sequencing is an efficient tool for variant late-infantile neuronal ceroid lipofuscinosis molecular diagnosis.

PubMed

Patiño, Liliana Catherine; Battu, Rajani; Ortega-Recalde, Oscar; Nallathambi, Jeyabalan; Anandula, Venkata Ramana; Renukaradhya, Umashankar; Laissue, Paul

2014-01-01

The neuronal ceroid-lipofuscinoses (NCL) is a group of neurodegenerative disorders characterized by epilepsy, visual failure, progressive mental and motor deterioration, myoclonus, dementia and reduced life expectancy. Classically, NCL-affected individuals have been classified into six categories, which have been mainly defined regarding the clinical onset of symptoms. However, some patients cannot be easily included in a specific group because of significant variation in the age of onset and disease progression. Molecular genetics has emerged in recent years as a useful tool for enhancing NCL subtype classification. Fourteen NCL genetic forms (CLN1 to CLN14) have been described to date. The variant late-infantile form of the disease has been linked to CLN5, CLN6, CLN7 (MFSD8) and CLN8 mutations. Despite advances in the diagnosis of neurodegenerative disorders mutations in these genes may cause similar phenotypes, which rends difficult accurate candidate gene selection for direct sequencing. Three siblings who were affected by variant late-infantile NCL are reported in the present study. We used whole-exome sequencing, direct sequencing and in silico approaches to identify the molecular basis of the disease. We identified the novel c.1219T>C (p.Trp407Arg) and c.1361T>C (p.Met454Thr) MFSD8 pathogenic mutations. Our results highlighted next generation sequencing as a novel and powerful methodological approach for the rapid determination of the molecular diagnosis of NCL. They also provide information regarding the phenotypic and molecular spectrum of CLN7 disease.

Preparation of a Trp-BODIPY fluorogenic amino acid to label peptides for enhanced live-cell fluorescence imaging.

PubMed

Mendive-Tapia, Lorena; Subiros-Funosas, Ramon; Zhao, Can; Albericio, Fernando; Read, Nick D; Lavilla, Rodolfo; Vendrell, Marc

2017-08-01

Fluorescent peptides are valuable tools for live-cell imaging because of the high specificity of peptide sequences for their biomolecular targets. When preparing fluorescent versions of peptides, labels must be introduced at appropriate positions in the sequences to provide suitable reporters while avoiding any impairment of the molecular recognition properties of the peptides. This protocol describes the preparation of the tryptophan (Trp)-based fluorogenic amino acid Fmoc-Trp(C 2 -BODIPY)-OH and its incorporation into peptides for live-cell fluorescence imaging-an approach that is applicable to most peptide sequences. Fmoc-Trp(C 2 -BODIPY)-OH contains a BODIPY (4,4-difluoro-4-bora-3a,4a-diaza-s-indacene) fluorogenic core, which works as an environmentally sensitive fluorophore, showing high fluorescence in lipophilic conditions. It is attached to Trp via a spacer-free C-C linkage, resulting in a labeled amino acid that can mimic the molecular interactions of Trp, enabling wash-free imaging. This protocol covers the chemical synthesis of the fluorogenic amino acid Fmoc-Trp(C 2 -BODIPY)-OH (3-4 d), the preparation of the labeled antimicrobial peptide BODIPY-cPAF26 by solid-phase synthesis (6-7 d) and its spectral and biological characterization as a live-cell imaging probe for different fungal pathogens. As an example, we include a procedure for using BODIPY-cPAF26 for wash-free imaging of fungal pathogens, including real-time visualization of Aspergillus fumigatus (5 d for culturing, 1-2 d for imaging).

Phylogeny of flowering plants by the chloroplast genome sequences: in search of a "lucky gene".

PubMed

Logacheva, M D; Penin, A A; Samigullin, T H; Vallejo-Roman, C M; Antonov, A S

2007-12-01

One of the most complicated remaining problems of molecular-phylogenetic analysis is choosing an appropriate genome region. In an ideal case, such a region should have two specific properties: (i) results of analysis using this region should be similar to the results of multigene analysis using the maximal number of regions; (ii) this region should be arranged compactly and be significantly shorter than the multigene set. The second condition is necessary to facilitate sequencing and extension of taxons under analysis, the number of which is also crucial for molecular phylogenetic analysis. Such regions have been revealed for some groups of animals and have been designated as "lucky genes". We have carried out a computational experiment on analysis of 41 complete chloroplast genomes of flowering plants aimed at searching for a "lucky gene" for reconstruction of their phylogeny. It is shown that the phylogenetic tree inferred from a combination of translated nucleotide sequences of genes encoding subunits of plastid RNA polymerase is closest to the tree constructed using all protein coding sites of the chloroplast genome. The only node for which a contradiction is observed is unstable according to the different type analyses. For all the other genes or their combinations, the coincidence is significantly worse. The RNA polymerase genes are compactly arranged in the genome and are fourfold shorter than the total length of protein coding genes used for phylogenetic analysis. The combination of all necessary features makes this group of genes main candidates for the role of "lucky gene" in studying phylogeny of flowering plants.

Under-detection of endospore-forming Firmicutes in metagenomic data

DOE PAGES

Filippidou, Sevasti; Junier, Thomas; Wunderlin, Tina; ...

2015-04-25

Microbial diversity studies based on metagenomic sequencing have greatly enhanced our knowledge of the microbial world. However, one caveat is the fact that not all microorganisms are equally well detected, questioning the universality of this approach. Firmicutes are known to be a dominant bacterial group. Several Firmicutes species are endospore formers and this property makes them hardy in potentially harsh conditions, and thus likely to be present in a wide variety of environments, even as residents and not functional players. While metagenomic libraries can be expected to contain endospore formers, endospores are known to be resilient to many traditional methodsmore » of DNA isolation and thus potentially undetectable. In this study we evaluated the representation of endospore-forming Firmicutes in 73 published metagenomic datasets using two molecular markers unique to this bacterial group ( spo0A and gpr). Both markers were notably absent in well-known habitats of Firmicutes such as soil, with spo0A found only in three mammalian gut microbiomes. A tailored DNA extraction method resulted in the detection of a large diversity of endospore-formers in amplicon sequencing of the 16S rRNA and spo0A genes. However, shotgun classification was still poor with only a minor fraction of the community assigned to Firmicutes. Thus, removing a specific bias in a molecular workflow improves detection in amplicon sequencing, but it was insufficient to overcome the limitations for detecting endospore-forming Firmicutes in whole-genome metagenomics. In conclusion, this study highlights the importance of understanding the specific methodological biases that can contribute to improve the universality of metagenomic approaches.« less

Under-detection of endospore-forming Firmicutes in metagenomic data

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

Filippidou, Sevasti; Junier, Thomas; Wunderlin, Tina

Microbial diversity studies based on metagenomic sequencing have greatly enhanced our knowledge of the microbial world. However, one caveat is the fact that not all microorganisms are equally well detected, questioning the universality of this approach. Firmicutes are known to be a dominant bacterial group. Several Firmicutes species are endospore formers and this property makes them hardy in potentially harsh conditions, and thus likely to be present in a wide variety of environments, even as residents and not functional players. While metagenomic libraries can be expected to contain endospore formers, endospores are known to be resilient to many traditional methodsmore » of DNA isolation and thus potentially undetectable. In this study we evaluated the representation of endospore-forming Firmicutes in 73 published metagenomic datasets using two molecular markers unique to this bacterial group ( spo0A and gpr). Both markers were notably absent in well-known habitats of Firmicutes such as soil, with spo0A found only in three mammalian gut microbiomes. A tailored DNA extraction method resulted in the detection of a large diversity of endospore-formers in amplicon sequencing of the 16S rRNA and spo0A genes. However, shotgun classification was still poor with only a minor fraction of the community assigned to Firmicutes. Thus, removing a specific bias in a molecular workflow improves detection in amplicon sequencing, but it was insufficient to overcome the limitations for detecting endospore-forming Firmicutes in whole-genome metagenomics. In conclusion, this study highlights the importance of understanding the specific methodological biases that can contribute to improve the universality of metagenomic approaches.« less

Molecular and functional characterization of Toll-like receptor (Tlr)1 and Tlr2 in common carp (Cyprinus carpio).

PubMed

Fink, Inge R; Pietretti, Danilo; Voogdt, Carlos G P; Westphal, Adrie H; Savelkoul, Huub F J; Forlenza, Maria; Wiegertjes, Geert F

2016-09-01

Toll-like receptors (TLRs) are fundamental components of innate immunity that play significant roles in the defence against pathogen invasion. In this study, we present the molecular characterization of the full-length coding sequence of tlr1, tlr2a and tlr2b from common carp (Cyprinus carpio). Each is encoded within a single exon and contains a conserved number of leucine-rich repeats, a transmembrane region and an intracellular TIR domain for signalling. Indeed, sequence, phylogenetic and synteny analysis of carp tlr1, tlr2a and tlr2b support that these genes are orthologues of mammalian TLR1 and TLR2. The tlr genes are expressed in various immune organs and cell types. Furthermore, the carp sequences exhibited a good three-dimensional fit with the heterodimer structure of human TLR1-TLR2, including the potential to bind to the ligand Pam3CSK4. This supports the possible formation of carp Tlr1-Tlr2 heterodimers. However, we were unable to demonstrate Tlr1/Tlr2-mediated ligand binding in transfected cell lines through NF-κB activation, despite showing the expression and co-localization of Tlr1 and Tlr2. We discuss possible limitations when studying ligand-specific activation of NF-κB after expression of Tlr1 and/or Tlr2 in human but also fish cell lines and we propose alternative future strategies for studying ligand-binding properties of fish Tlrs. Copyright © 2016 The Authors. Published by Elsevier Ltd.. All rights reserved.

Single molecule counting and assessment of random molecular tagging errors with transposable giga-scale error-correcting barcodes.

PubMed

Lau, Billy T; Ji, Hanlee P

2017-09-21

RNA-Seq measures gene expression by counting sequence reads belonging to unique cDNA fragments. Molecular barcodes commonly in the form of random nucleotides were recently introduced to improve gene expression measures by detecting amplification duplicates, but are susceptible to errors generated during PCR and sequencing. This results in false positive counts, leading to inaccurate transcriptome quantification especially at low input and single-cell RNA amounts where the total number of molecules present is minuscule. To address this issue, we demonstrated the systematic identification of molecular species using transposable error-correcting barcodes that are exponentially expanded to tens of billions of unique labels. We experimentally showed random-mer molecular barcodes suffer from substantial and persistent errors that are difficult to resolve. To assess our method's performance, we applied it to the analysis of known reference RNA standards. By including an inline random-mer molecular barcode, we systematically characterized the presence of sequence errors in random-mer molecular barcodes. We observed that such errors are extensive and become more dominant at low input amounts. We described the first study to use transposable molecular barcodes and its use for studying random-mer molecular barcode errors. Extensive errors found in random-mer molecular barcodes may warrant the use of error correcting barcodes for transcriptome analysis as input amounts decrease.

Screening, Characterization and In Vitro Evaluation of Probiotic Properties Among Lactic Acid Bacteria Through Comparative Analysis.

PubMed

Devi, Sundru Manjulata; Archer, Ann Catherine; Halami, Prakash M

2015-09-01

The present work aimed to identify probiotic bacteria from healthy human infant faecal and dairy samples. Subsequently, an assay was developed to evaluate the probiotic properties using comparative genetic approach for marker genes involved in adhesion to the intestinal epithelial layer. Several in vitro properties including tolerance to biological barriers (such as acid and bile), antimicrobial spectrum, resistance to simulated digestive fluids and cellular hydrophobicity were assessed. The potential probiotic cultures were rapidly characterized by morphological, physiological and molecular-based methods [such as RFLP, ITS, RAPD and (GTG)5]. Further analysis by 16S rDNA sequencing revealed that the selected isolates belong to Lactobacillus, Pediococcus and Enterococcus species. Two cultures of non-lactic, non-pathogenic Staphylococcus spp. were also isolated. The native isolates were able to survive under acidic, bile and simulated intestinal conditions. In addition, these cultures inhibited the growth of tested bacterial pathogens. Further, no correlation was observed between hydrophobicity and adhesion ability. Sequencing of probiotic marker genes such as bile salt hydrolase (bsh), fibronectin-binding protein (fbp) and mucin-binding protein (mub) for selected isolates revealed nucleotide variation. The probiotic binding domains were detected by several bioinformatic tools. The approach used in the study enabled the identification of potential probiotic domains responsible for adhesion of bacteria to intestinal epithelial layer, which may further assist in screening of novel probiotic bacteria. The rapid detection of binding domains will help in revealing the beneficial properties of the probiotic cultures. Further, studies will be performed to develop a novel probiotic product which will contribute in food and feed industry.

Detecting very low allele fraction variants using targeted DNA sequencing and a novel molecular barcode-aware variant caller.

PubMed

Xu, Chang; Nezami Ranjbar, Mohammad R; Wu, Zhong; DiCarlo, John; Wang, Yexun

2017-01-03

Detection of DNA mutations at very low allele fractions with high accuracy will significantly improve the effectiveness of precision medicine for cancer patients. To achieve this goal through next generation sequencing, researchers need a detection method that 1) captures rare mutation-containing DNA fragments efficiently in the mix of abundant wild-type DNA; 2) sequences the DNA library extensively to deep coverage; and 3) distinguishes low level true variants from amplification and sequencing errors with high accuracy. Targeted enrichment using PCR primers provides researchers with a convenient way to achieve deep sequencing for a small, yet most relevant region using benchtop sequencers. Molecular barcoding (or indexing) provides a unique solution for reducing sequencing artifacts analytically. Although different molecular barcoding schemes have been reported in recent literature, most variant calling has been done on limited targets, using simple custom scripts. The analytical performance of barcode-aware variant calling can be significantly improved by incorporating advanced statistical models. We present here a highly efficient, simple and scalable enrichment protocol that integrates molecular barcodes in multiplex PCR amplification. In addition, we developed smCounter, an open source, generic, barcode-aware variant caller based on a Bayesian probabilistic model. smCounter was optimized and benchmarked on two independent read sets with SNVs and indels at 5 and 1% allele fractions. Variants were called with very good sensitivity and specificity within coding regions. We demonstrated that we can accurately detect somatic mutations with allele fractions as low as 1% in coding regions using our enrichment protocol and variant caller.

Molecular and Physiological Analysis of a Heat-Shock Response in Wheat 1

PubMed Central

McElwain, Elizabeth F.; Spiker, Steven

1992-01-01

We have isolated two cDNA clones from wheat (Triticum aestivum L. var Stephens), designated WHSP16.8 and WHSP16.9, that are highly similar in sequence to the low molecular weight heat-shock protein genes previously isolated from soybean. RNA blot analysis confirms that these sequences are present in heat-shocked wheat seedlings, but not in control tissues. The WHSP16.8 and WHSP16.9 cDNAs were isolated by screening a lambda gt11 expression library with antibodies to HMGc (a chromosomal protein of wheat). Immunoblot analysis has demonstrated that the antibodies raised against HMGc also recognize a group of proteins that are induced by heat shock and have molecular weights (estimated by sodium dodecyl sulfate electrophoresis) consistent with the molecular weights of the proteins deduced from the sequences of the cDNAs. ImagesFigure 3Figure 4Figure 5 PMID:16669058

Managing the genomic revolution in cancer diagnostics.

PubMed

Nguyen, Doreen; Gocke, Christopher D

2017-08-01

Molecular tumor profiling is now a routine part of patient care, revealing targetable genomic alterations and molecularly distinct tumor subtypes with therapeutic and prognostic implications. The widespread adoption of next-generation sequencing technologies has greatly facilitated clinical implementation of genomic data and opened the door for high-throughput multigene-targeted sequencing. Herein, we discuss the variability of cancer genetic profiling currently offered by clinical laboratories, the challenges of applying rapidly evolving medical knowledge to individual patients, and the need for more standardized population-based molecular profiling.

New concepts for molecular magnets

NASA Astrophysics Data System (ADS)

Pilawa, Bernd

1999-03-01

Miller and Epstein (1994) define molecular magnets as magnetic materials which are prepared by the low-temperature methods of the preparative chemistry. This definition includes molecular crystals of neutral radicals, radical salts and charge transfer complexes as well as metal complexes and polymers with unpaired spins (Dormann 1995). The challenge of molecular magnets consists in tailoring magnetic properties by specific modifications of the molecular units. The combination of magnetism with mechanical or electrical properties of molecular compounds promise materials of high technical interest (Gatteschi 1994a and 1994b, Möhwald 1996) and both the chemical synthesis of new molecular materials with magnetic properties as well as the physical investigation and explanation of these properties is important, in order to achieve any progress. This work deals with the physical characterization of the magnetic properties of molecular materials. It is organized as follows. In the first part molecular crystals of neutral radicals are studied. After briefly discussing the general magnetic properties of these materials and after an overview over the physical principles of exchange interaction between organic radicals I focus on the interplay between the crystallographic structure and the magnetic properties of various derivatives of the verdazyl and nitronyl nitroxide radicals. The magnetic properties of metal complexes are the subject of the second part. After an overview over the experimental and theoretical tools which are used for the investigation of the magnetic properties I shortly discuss the exchange coupling of transition metal ions and the magnetic properties of complexes of two and three metal ions. Special emphasis is given to spin cluster compounds. Spin cluster denote complexes of many magnetic ions. They are attractive as building blocks of molecular magnets as well as magnetic model compounds for the study of spin frustration, molecular super-paramagnetism and quasi one-dimensional magnets.

The efficacy of microarray screening for autosomal recessive retinitis pigmentosa in routine clinical practice

PubMed Central

van Huet, Ramon A. C.; Pierrache, Laurence H.M.; Meester-Smoor, Magda A.; Klaver, Caroline C.W.; van den Born, L. Ingeborgh; Hoyng, Carel B.; de Wijs, Ilse J.; Collin, Rob W. J.; Hoefsloot, Lies H.

2015-01-01

Purpose To determine the efficacy of multiple versions of a commercially available arrayed primer extension (APEX) microarray chip for autosomal recessive retinitis pigmentosa (arRP). Methods We included 250 probands suspected of arRP who were genetically analyzed with the APEX microarray between January 2008 and November 2013. The mode of inheritance had to be autosomal recessive according to the pedigree (including isolated cases). If the microarray identified a heterozygous mutation, we performed Sanger sequencing of exons and exon–intron boundaries of that specific gene. The efficacy of this microarray chip with the additional Sanger sequencing approach was determined by the percentage of patients that received a molecular diagnosis. We also collected data from genetic tests other than the APEX analysis for arRP to provide a detailed description of the molecular diagnoses in our study cohort. Results The APEX microarray chip for arRP identified the molecular diagnosis in 21 (8.5%) of the patients in our cohort. Additional Sanger sequencing yielded a second mutation in 17 patients (6.8%), thereby establishing the molecular diagnosis. In total, 38 patients (15.2%) received a molecular diagnosis after analysis using the microarray and additional Sanger sequencing approach. Further genetic analyses after a negative result of the arRP microarray (n = 107) resulted in a molecular diagnosis of arRP (n = 23), autosomal dominant RP (n = 5), X-linked RP (n = 2), and choroideremia (n = 1). Conclusions The efficacy of the commercially available APEX microarray chips for arRP appears to be low, most likely caused by the limitations of this technique and the genetic and allelic heterogeneity of RP. Diagnostic yields up to 40% have been reported for next-generation sequencing (NGS) techniques that, as expected, thereby outperform targeted APEX analysis. PMID:25999674

Molecular taxonomy of phytopathogenic fungi: a case study in Peronospora.

PubMed

Göker, Markus; García-Blázquez, Gema; Voglmayr, Hermann; Tellería, M Teresa; Martín, María P

2009-07-29

Inappropriate taxon definitions may have severe consequences in many areas. For instance, biologically sensible species delimitation of plant pathogens is crucial for measures such as plant protection or biological control and for comparative studies involving model organisms. However, delimiting species is challenging in the case of organisms for which often only molecular data are available, such as prokaryotes, fungi, and many unicellular eukaryotes. Even in the case of organisms with well-established morphological characteristics, molecular taxonomy is often necessary to emend current taxonomic concepts and to analyze DNA sequences directly sampled from the environment. Typically, for this purpose clustering approaches to delineate molecular operational taxonomic units have been applied using arbitrary choices regarding the distance threshold values, and the clustering algorithms. Here, we report on a clustering optimization method to establish a molecular taxonomy of Peronospora based on ITS nrDNA sequences. Peronospora is the largest genus within the downy mildews, which are obligate parasites of higher plants, and includes various economically important pathogens. The method determines the distance function and clustering setting that result in an optimal agreement with selected reference data. Optimization was based on both taxonomy-based and host-based reference information, yielding the same outcome. Resampling and permutation methods indicate that the method is robust regarding taxon sampling and errors in the reference data. Tests with newly obtained ITS sequences demonstrate the use of the re-classified dataset in molecular identification of downy mildews. A corrected taxonomy is provided for all Peronospora ITS sequences contained in public databases. Clustering optimization appears to be broadly applicable in automated, sequence-based taxonomy. The method connects traditional and modern taxonomic disciplines by specifically addressing the issue of how to optimally account for both traditional species concepts and genetic divergence.

Molecular Taxonomy of Phytopathogenic Fungi: A Case Study in Peronospora

PubMed Central

Göker, Markus; García-Blázquez, Gema; Voglmayr, Hermann; Tellería, M. Teresa; Martín, María P.

2009-01-01

Background Inappropriate taxon definitions may have severe consequences in many areas. For instance, biologically sensible species delimitation of plant pathogens is crucial for measures such as plant protection or biological control and for comparative studies involving model organisms. However, delimiting species is challenging in the case of organisms for which often only molecular data are available, such as prokaryotes, fungi, and many unicellular eukaryotes. Even in the case of organisms with well-established morphological characteristics, molecular taxonomy is often necessary to emend current taxonomic concepts and to analyze DNA sequences directly sampled from the environment. Typically, for this purpose clustering approaches to delineate molecular operational taxonomic units have been applied using arbitrary choices regarding the distance threshold values, and the clustering algorithms. Methodology Here, we report on a clustering optimization method to establish a molecular taxonomy of Peronospora based on ITS nrDNA sequences. Peronospora is the largest genus within the downy mildews, which are obligate parasites of higher plants, and includes various economically important pathogens. The method determines the distance function and clustering setting that result in an optimal agreement with selected reference data. Optimization was based on both taxonomy-based and host-based reference information, yielding the same outcome. Resampling and permutation methods indicate that the method is robust regarding taxon sampling and errors in the reference data. Tests with newly obtained ITS sequences demonstrate the use of the re-classified dataset in molecular identification of downy mildews. Conclusions A corrected taxonomy is provided for all Peronospora ITS sequences contained in public databases. Clustering optimization appears to be broadly applicable in automated, sequence-based taxonomy. The method connects traditional and modern taxonomic disciplines by specifically addressing the issue of how to optimally account for both traditional species concepts and genetic divergence. PMID:19641601

Toolbox Approaches Using Molecular Markers and 16S rRNA Gene Amplicon Data Sets for Identification of Fecal Pollution in Surface Water.

PubMed

Ahmed, W; Staley, C; Sadowsky, M J; Gyawali, P; Sidhu, J P S; Palmer, A; Beale, D J; Toze, S

2015-10-01

In this study, host-associated molecular markers and bacterial 16S rRNA gene community analysis using high-throughput sequencing were used to identify the sources of fecal pollution in environmental waters in Brisbane, Australia. A total of 92 fecal and composite wastewater samples were collected from different host groups (cat, cattle, dog, horse, human, and kangaroo), and 18 water samples were collected from six sites (BR1 to BR6) along the Brisbane River in Queensland, Australia. Bacterial communities in the fecal, wastewater, and river water samples were sequenced. Water samples were also tested for the presence of bird-associated (GFD), cattle-associated (CowM3), horse-associated, and human-associated (HF183) molecular markers, to provide multiple lines of evidence regarding the possible presence of fecal pollution associated with specific hosts. Among the 18 water samples tested, 83%, 33%, 17%, and 17% were real-time PCR positive for the GFD, HF183, CowM3, and horse markers, respectively. Among the potential sources of fecal pollution in water samples from the river, DNA sequencing tended to show relatively small contributions from wastewater treatment plants (up to 13% of sequence reads). Contributions from other animal sources were rarely detected and were very small (<3% of sequence reads). Source contributions determined via sequence analysis versus detection of molecular markers showed variable agreement. A lack of relationships among fecal indicator bacteria, host-associated molecular markers, and 16S rRNA gene community analysis data was also observed. Nonetheless, we show that bacterial community and host-associated molecular marker analyses can be combined to identify potential sources of fecal pollution in an urban river. This study is a proof of concept, and based on the results, we recommend using bacterial community analysis (where possible) along with PCR detection or quantification of host-associated molecular markers to provide information on the sources of fecal pollution in waterways. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

Molecular Profiling of Malignant Pleural Effusion in Metastatic Non-Small-Cell Lung Carcinoma. The Effect of Preanalytical Factors.

PubMed

Carter, Jamal; Miller, James Adam; Feller-Kopman, David; Ettinger, David; Sidransky, David; Maleki, Zahra

2017-07-01

Non-small-cell lung cancer (NSCLC)-associated malignant pleural effusions (MPEs) are sometimes the only available specimens for molecular analysis. This study evaluates diagnostic yield of NSCLC-associated MPE, its adequacy for molecular profiling and the potential influence of MPE volume/cellularity on the analytic sensitivity of our assays. Molecular results of 50 NSCLC-associated MPE cases during a 5-year period were evaluated. Molecular profiling was performed on cell blocks and consisted of fluorescent in situ hybridization (FISH) for ALK gene rearrangements and the following sequencing platforms: Sanger sequencing (for EGFR) and high-throughput pyrosequencing (for KRAS and BRAF) during the first 4 years of the study period, and targeted next-generation sequencing performed thereafter. A total of 50 NSCLC-associated MPE cases were identified where molecular testing was requested. Of these, 17 cases were excluded: 14 cases (28%) due to inadequate tumor cellularity and 3 cases due to unavailability of the slides to review. A total of 27 out of 50 MPE cases (54%) underwent at least EGFR and KRAS sequencing and FISH for ALK rearrangement. Of the 27 cases with molecular testing results available, a genetic abnormality was detected in 16 cases (59%). The most common genetic aberrations identified involved EGFR ( 9 ) and KRAS ( 7 ). Six cases had ALK FISH only, of which one showed rearrangement. MPE volume was not associated with overall cellularity or tumor cellularity (P = 0.360). Molecular profiling of MPE is a viable alternative to testing solid tissue in NSCLC. This study shows successful detection of genetic aberrations in 59% of samples with minimal risk of false negative.

Type II restriction endonucleases—a historical perspective and more

PubMed Central

Pingoud, Alfred; Wilson, Geoffrey G.; Wende, Wolfgang

2014-01-01

This article continues the series of Surveys and Summaries on restriction endonucleases (REases) begun this year in Nucleic Acids Research. Here we discuss ‘Type II’ REases, the kind used for DNA analysis and cloning. We focus on their biochemistry: what they are, what they do, and how they do it. Type II REases are produced by prokaryotes to combat bacteriophages. With extreme accuracy, each recognizes a particular sequence in double-stranded DNA and cleaves at a fixed position within or nearby. The discoveries of these enzymes in the 1970s, and of the uses to which they could be put, have since impacted every corner of the life sciences. They became the enabling tools of molecular biology, genetics and biotechnology, and made analysis at the most fundamental levels routine. Hundreds of different REases have been discovered and are available commercially. Their genes have been cloned, sequenced and overexpressed. Most have been characterized to some extent, but few have been studied in depth. Here, we describe the original discoveries in this field, and the properties of the first Type II REases investigated. We discuss the mechanisms of sequence recognition and catalysis, and the varied oligomeric modes in which Type II REases act. We describe the surprising heterogeneity revealed by comparisons of their sequences and structures. PMID:24878924

Phenotypic and genomic analysis of serotype 3 Sabin poliovirus vaccine produced in MRC-5 cell substrate.

PubMed

Alirezaie, Behnam; Taqavian, Mohammad; Aghaiypour, Khosrow; Esna-Ashari, Fatemeh; Shafyi, Abbas

2011-05-01

The cell substrate has a pivotal role in live virus vaccines production. It is necessary to evaluate the effects of the cell substrate on the properties of the propagated viruses, especially in the case of viruses which are unstable genetically such as polioviruses, by monitoring the molecular and phenotypical characteristics of harvested viruses. To investigate the presence/absence of mutation(s), the near full-length genomic sequence of different harvests of the type 3 Sabin strain of poliovirus propagated in MRC-5 cells were determined. The sequences were compared with genomic sequences of different virus seeds, vaccines, and OPV-like isolates. Nearly complete genomic sequencing results, however, revealed no detectable mutations throughout the genome RNA-plaque purified (RSO)-derived monopool of type 3 OPVs manufactured in MRC-5. Thirty-six years of experience in OPV production, trend analysis, and vaccine surveillance also suggest that: (i) different monopools of serotype 3 OPV produced in MRC-5 retained their phenotypic characteristics (temperature sensitivity and neuroattenuation), (ii) MRC-5 cells support the production of acceptable virus yields, (iii) OPV replicated in the MRC-5 cell substrate is a highly efficient and safe vaccine. These results confirm previous reports that MRC-5 is a desirable cell substrate for the production of OPV. Copyright © 2011 Wiley-Liss, Inc.

Protein and gene structure of a blue laccase from Pleurotus ostreatus1.

PubMed Central

Giardina, P; Palmieri, G; Scaloni, A; Fontanella, B; Faraco, V; Cennamo, G; Sannia, G

1999-01-01

A new laccase isoenzyme (POXA1b, where POX is phenol oxidase), produced by Pleurotus ostreatus in cultures supplemented with copper sulphate, has been purified and fully characterized. The main characteristics of this protein (molecular mass in native and denaturing conditions, pI and catalytic properties) are almost identical to the previously studied laccase POXA1w. However, POXA1b contains four copper atoms per molecule instead of one copper, two zinc and one iron atom per molecule of POXA1w. Furthermore, POXA1b shows an unusually high stability at alkaline pH. The gene and cDNA coding for POXA1b have been cloned and sequenced. The gene coding sequence contains 1599 bp, interrupted by 15 introns. Comparison of the structure of the poxa1b gene with the two previously studied P. ostreatus laccase genes (pox1 and poxc) suggests that these genes belong to two different subfamilies. The amino acid sequence of POXA1b deduced from the cDNA sequence has been almost completely verified by means of matrix-assisted laser desorption ionization MS. It has been demonstrated that three out of six putative glycosylation sites are post-translationally modified and the structure of the bound glycosidic moieties has been determined, whereas two other putative glycosylation sites are unmodified. PMID:10417329

Biochemical and Molecular Phylogenetic Study of Agriculturally Useful Association of a Nitrogen-Fixing Cyanobacterium and Nodule Sinorhizobium with Medicago sativa L.

PubMed Central

Karaushu, E. V.; Kravzova, T. R.; Vorobey, N. A.; Kiriziy, D. A.; Olkhovich, O. P.; Taran, N. Yu.; Kots, S. Ya.; Omarova, E.

2015-01-01

Seed inoculation with bacterial consortium was found to increase legume yield, providing a higher growth than the standard nitrogen treatment methods. Alfalfa plants were inoculated by mono- and binary compositions of nitrogen-fixing microorganisms. Their physiological and biochemical properties were estimated. Inoculation by microbial consortium of Sinorhizobium meliloti T17 together with a new cyanobacterial isolate Nostoc PTV was more efficient than the single-rhizobium strain inoculation. This treatment provides an intensification of the processes of biological nitrogen fixation by rhizobia bacteria in the root nodules and an intensification of plant photosynthesis. Inoculation by bacterial consortium stimulates growth of plant mass and rhizogenesis and leads to increased productivity of alfalfa and to improving the amino acid composition of plant leaves. The full nucleotide sequence of the rRNA gene cluster and partial sequence of the dinitrogenase reductase (nifH) gene of Nostoc PTV were deposited to GenBank (JQ259185.1, JQ259186.1). Comparison of these gene sequences of Nostoc PTV with all sequences present at the GenBank shows that this cyanobacterial strain does not have 100% identity with any organisms investigated previously. Phylogenetic analysis showed that this cyanobacterium clustered with high credibility values with Nostoc muscorum. PMID:26114100

Biochemical and Molecular Phylogenetic Study of Agriculturally Useful Association of a Nitrogen-Fixing Cyanobacterium and Nodule Sinorhizobium with Medicago sativa L.

PubMed

Karaushu, E V; Lazebnaya, I V; Kravzova, T R; Vorobey, N A; Lazebny, O E; Kiriziy, D A; Olkhovich, O P; Taran, N Yu; Kots, S Ya; Popova, A A; Omarova, E; Koksharova, O A

2015-01-01

Seed inoculation with bacterial consortium was found to increase legume yield, providing a higher growth than the standard nitrogen treatment methods. Alfalfa plants were inoculated by mono- and binary compositions of nitrogen-fixing microorganisms. Their physiological and biochemical properties were estimated. Inoculation by microbial consortium of Sinorhizobium meliloti T17 together with a new cyanobacterial isolate Nostoc PTV was more efficient than the single-rhizobium strain inoculation. This treatment provides an intensification of the processes of biological nitrogen fixation by rhizobia bacteria in the root nodules and an intensification of plant photosynthesis. Inoculation by bacterial consortium stimulates growth of plant mass and rhizogenesis and leads to increased productivity of alfalfa and to improving the amino acid composition of plant leaves. The full nucleotide sequence of the rRNA gene cluster and partial sequence of the dinitrogenase reductase (nifH) gene of Nostoc PTV were deposited to GenBank (JQ259185.1, JQ259186.1). Comparison of these gene sequences of Nostoc PTV with all sequences present at the GenBank shows that this cyanobacterial strain does not have 100% identity with any organisms investigated previously. Phylogenetic analysis showed that this cyanobacterium clustered with high credibility values with Nostoc muscorum.

EffectorP: predicting fungal effector proteins from secretomes using machine learning.

PubMed

Sperschneider, Jana; Gardiner, Donald M; Dodds, Peter N; Tini, Francesco; Covarelli, Lorenzo; Singh, Karam B; Manners, John M; Taylor, Jennifer M

2016-04-01

Eukaryotic filamentous plant pathogens secrete effector proteins that modulate the host cell to facilitate infection. Computational effector candidate identification and subsequent functional characterization delivers valuable insights into plant-pathogen interactions. However, effector prediction in fungi has been challenging due to a lack of unifying sequence features such as conserved N-terminal sequence motifs. Fungal effectors are commonly predicted from secretomes based on criteria such as small size and cysteine-rich, which suffers from poor accuracy. We present EffectorP which pioneers the application of machine learning to fungal effector prediction. EffectorP improves fungal effector prediction from secretomes based on a robust signal of sequence-derived properties, achieving sensitivity and specificity of over 80%. Features that discriminate fungal effectors from secreted noneffectors are predominantly sequence length, molecular weight and protein net charge, as well as cysteine, serine and tryptophan content. We demonstrate that EffectorP is powerful when combined with in planta expression data for predicting high-priority effector candidates. EffectorP is the first prediction program for fungal effectors based on machine learning. Our findings will facilitate functional fungal effector studies and improve our understanding of effectors in plant-pathogen interactions. EffectorP is available at http://effectorp.csiro.au. © 2015 CSIRO New Phytologist © 2015 New Phytologist Trust.

Molecular cloning and transcriptional analysis of a NPY receptor-like in common Chinese cuttlefish Sepiella japonica

NASA Astrophysics Data System (ADS)

Yang, Jingwen; Xu, Yuchao; Xu, Ke; Ping, Hongling; Shi, Huilai; Lü, Zhenming; Wu, Changwen; Wang, Tianming

2017-08-01

Neuropeptide Y (NPY) has a pivotal role in the regulation of many physiological processes. In this study, the gene encoding a NPY receptor-like from the common Chinese cuttlefish Sepiella japonica (SjNPYR-like) was identified and characterized. The full-length SjNPYR-like cDNA was cloned containing a 492-bp of 5' untranslated region (UTR), 1 182 bp open reading frame (ORF) encoding a protein of 393 amino acid residues, and 228 bp of 3' UTR. The putative protein was predicted to have a molecular weight of 45.54 kDa and an isoelectric point (pI) of 8.13. By informatic analyses, SjNPYR-like was identified as belonging to the class A G protein coupled receptor (GPCR) family (the rhodopsin-type). The amino acid sequence contained 12 potential phosphorylation sites and five predicted N-linked glycosylation sites. Multiple sequence alignment and 3D structure modeling were conducted to clarify SjNPYR bioinformatics characteristics. Phylogenetic analysis identifies it as an NPYR with identity of 33% to Lymnaea stagnalis NPFR. Transmembrane properties of SjNPYR-like were demonstrated in vitro using HEK293 cells and the pEGFP-N1 plasmid. Relative quantification of SjNPYR-like mRNA level confirmed a high level expression and broad distribution of SjNPYR - like in various tissues of female S. japonica. In addition, the transcriptional profile of SjNPYR - like in the brain, liver, and ovary during gonadal development was analyzed. The results provide basic understanding on the molecular characteristics of SjNPYR-like and its potentially physical functions.

Peptoid conformational free energy landscapes from implicit-solvent molecular simulations in AMBER.

PubMed

Voelz, Vincent A; Dill, Ken A; Chorny, Ilya

2011-01-01

To test the accuracy of existing AMBER force field models in predicting peptoid conformation and dynamics, we simulated a set of model peptoid molecules recently examined by Butterfoss et al. (JACS 2009, 131, 16798-16807) using QM methods as well as three peptoid sequences with experimentally determined structures. We found that AMBER force fields, when used with a Generalized Born/Surface Area (GBSA) implicit solvation model, could accurately reproduce the peptoid torsional landscape as well as the major conformers of known peptoid structures. Enhanced sampling by replica exchange molecular dynamics (REMD) using temperatures from 300 to 800 K was used to sample over cis-trans isomerization barriers. Compared to (Nrch)5 and cyclo-octasarcosyl, the free energy of N-(2-nitro-3-hydroxyl phenyl)glycine-N-(phenyl)glycine has the most "foldable" free energy landscape, due to deep trans-amide minima dictated by N-aryl sidechains. For peptoids with (S)-N (1-phenylethyl) (Nspe) side chains, we observe a discrepancy in backbone dihedral propensities between molecular simulations and QM calculations, which may be due to force field effects or the inability to capture n --> n* interactions. For these residues, an empirical phi-angle biasing potential can "rescue" the backbone propensities seen in QM. This approach can serve as a general strategy for addressing force fields without resorting to a complete reparameterization. Overall, this study demonstrates the utility of implicit-solvent REMD simulations for efficient sampling to predict peptoid conformational landscapes, providing a potential tool for first-principles design of sequences with specific folding properties.

Long-range correlations and charge transport properties of DNA sequences

NASA Astrophysics Data System (ADS)

Liu, Xiao-liang; Ren, Yi; Xie, Qiong-tao; Deng, Chao-sheng; Xu, Hui

2010-04-01

By using Hurst's analysis and transfer approach, the rescaled range functions and Hurst exponents of human chromosome 22 and enterobacteria phage lambda DNA sequences are investigated and the transmission coefficients, Landauer resistances and Lyapunov coefficients of finite segments based on above genomic DNA sequences are calculated. In a comparison with quasiperiodic and random artificial DNA sequences, we find that λ-DNA exhibits anticorrelation behavior characterized by a Hurst exponent 0.5

Production And Characterization Of Synthetic Spider Silks Based On Nephila Clavipes Major Ampullate Silk Proteins

NASA Astrophysics Data System (ADS)

An, Bo

The extraordinary mechanical properties of orb-weaving spider silks have served spiders for over 400 million years. However, only in the late 20th century did we start to understand the molecular nature of spider silk that contributes to its incredible properties as biomaterials. Among all seven types of spider silks, major ampullate silk from typical orb-weaving spiders is the toughest of all, it consists of primarily two proteins: MaSp1 and MaSp2. Variable ratios and conserved motifs of these two proteins in all the native spider silks demonstrate the significant role of MaSp1 and MaSp2 in controlling the mechanical properties of the fiber. The amino acid sequences of the orb weaving spider silk proteins have remained almost unchanged for more than 100 million years. Interestingly, MaSp1 and MaSp2 are the only two components in all studied dragline silk fibers from these spiders. The mechanical properties of native dragline silk vary slightly between species, which are believed to relate to the ratio of MaSp1 to MaSp2 in the silk. Both of these facts clearly indicate the importance of these two proteins to the mechanical properties of the fiber. Various types of synthetic spider silk fibers have been produced and studied in an effort to mass-produce man-made fibers with qualities comparable to native spider silk. To investigate the roles of MaSp1 and MaSp2 in silk fiber, synthetic MaSp1 (major abundant protein in Nephila clavipes major ampullate silks) only fibers, MaSp1/MaSp2 protein mixture fibers and chimeric protein fibers with both MaSp1 and MaSp2 sequence features have been produced and tested for mechanical properties. Solid-State Nuclear Magnetic Resonance was used to characterize the structure of silk fibers and reveal the relation between fiber spatial structure and mechanical properties.

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

PubMed

Beyer, Stefan; Simmel, Friedrich C

2006-01-01

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

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

PubMed Central

Beyer, Stefan; Simmel, Friedrich C.

2006-01-01

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

`Inter-Arrival Time' Inspired Algorithm and its Application in Clustering and Molecular Phylogeny

NASA Astrophysics Data System (ADS)

Kolekar, Pandurang S.; Kale, Mohan M.; Kulkarni-Kale, Urmila

2010-10-01

Bioinformatics, being multidisciplinary field, involves applications of various methods from allied areas of Science for data mining using computational approaches. Clustering and molecular phylogeny is one of the key areas in Bioinformatics, which help in study of classification and evolution of organisms. Molecular phylogeny algorithms can be divided into distance based and character based methods. But most of these methods are dependent on pre-alignment of sequences and become computationally intensive with increase in size of data and hence demand alternative efficient approaches. `Inter arrival time distribution' (IATD) is a popular concept in the theory of stochastic system modeling but its potential in molecular data analysis has not been fully explored. The present study reports application of IATD in Bioinformatics for clustering and molecular phylogeny. The proposed method provides IATDs of nucleotides in genomic sequences. The distance function based on statistical parameters of IATDs is proposed and distance matrix thus obtained is used for the purpose of clustering and molecular phylogeny. The method is applied on a dataset of 3' non-coding region sequences (NCR) of Dengue virus type 3 (DENV-3), subtype III, reported in 2008. The phylogram thus obtained revealed the geographical distribution of DENV-3 isolates. Sri Lankan DENV-3 isolates were further observed to be clustered in two sub-clades corresponding to pre and post Dengue hemorrhagic fever emergence groups. These results are consistent with those reported earlier, which are obtained using pre-aligned sequence data as an input. These findings encourage applications of the IATD based method in molecular phylogenetic analysis in particular and data mining in general.

A Linked Series of Laboratory Exercises in Molecular Biology Utilizing Bioinformatics and GFP

ERIC Educational Resources Information Center

Medin, Carey L.; Nolin, Katie L.

2011-01-01

Molecular biologists commonly use bioinformatics to map and analyze DNA and protein sequences and to align different DNA and protein sequences for comparison. Additionally, biologists can create and view 3D models of protein structures to further understand intramolecular interactions. The primary goal of this 10-week laboratory was to introduce…

Thermoelectric effect and its dependence on molecular length and sequence in single DNA molecules.

PubMed

Li, Yueqi; Xiang, Limin; Palma, Julio L; Asai, Yoshihiro; Tao, Nongjian

2016-04-15

Studying the thermoelectric effect in DNA is important for unravelling charge transport mechanisms and for developing relevant applications of DNA molecules. Here we report a study of the thermoelectric effect in single DNA molecules. By varying the molecular length and sequence, we tune the charge transport in DNA to either a hopping- or tunnelling-dominated regimes. The thermoelectric effect is small and insensitive to the molecular length in the hopping regime. In contrast, the thermoelectric effect is large and sensitive to the length in the tunnelling regime. These findings indicate that one may control the thermoelectric effect in DNA by varying its sequence and length. We describe the experimental results in terms of hopping and tunnelling charge transport models.

Thermoelectric effect and its dependence on molecular length and sequence in single DNA molecules

PubMed Central

Li, Yueqi; Xiang, Limin; Palma, Julio L.; Asai, Yoshihiro; Tao, Nongjian

2016-01-01

Studying the thermoelectric effect in DNA is important for unravelling charge transport mechanisms and for developing relevant applications of DNA molecules. Here we report a study of the thermoelectric effect in single DNA molecules. By varying the molecular length and sequence, we tune the charge transport in DNA to either a hopping- or tunnelling-dominated regimes. The thermoelectric effect is small and insensitive to the molecular length in the hopping regime. In contrast, the thermoelectric effect is large and sensitive to the length in the tunnelling regime. These findings indicate that one may control the thermoelectric effect in DNA by varying its sequence and length. We describe the experimental results in terms of hopping and tunnelling charge transport models. PMID:27079152

A blackberry (Rubus L.) expressed sequence tag library for the development of simple sequence repeat markers

PubMed Central

Lewers, Kim S; Saski, Chris A; Cuthbertson, Brandon J; Henry, David C; Staton, Meg E; Main, Dorrie S; Dhanaraj, Anik L; Rowland, Lisa J; Tomkins, Jeff P

2008-01-01

Background The recent development of novel repeat-fruiting types of blackberry (Rubus L.) cultivars, combined with a long history of morphological marker-assisted selection for thornlessness by blackberry breeders, has given rise to increased interest in using molecular markers to facilitate blackberry breeding. Yet no genetic maps, molecular markers, or even sequences exist specifically for cultivated blackberry. The purpose of this study is to begin development of these tools by generating and annotating the first blackberry expressed sequence tag (EST) library, designing primers from the ESTs to amplify regions containing simple sequence repeats (SSR), and testing the usefulness of a subset of the EST-SSRs with two blackberry cultivars. Results A cDNA library of 18,432 clones was generated from expanding leaf tissue of the cultivar Merton Thornless, a progenitor of many thornless commercial cultivars. Among the most abundantly expressed of the 3,000 genes annotated were those involved with energy, cell structure, and defense. From individual sequences containing SSRs, 673 primer pairs were designed. Of a randomly chosen set of 33 primer pairs tested with two blackberry cultivars, 10 detected an average of 1.9 polymorphic PCR products. Conclusion This rate predicts that this library may yield as many as 940 SSR primer pairs detecting 1,786 polymorphisms. This may be sufficient to generate a genetic map that can be used to associate molecular markers with phenotypic traits, making possible molecular marker-assisted breeding to compliment existing morphological marker-assisted breeding in blackberry. PMID:18570660

A Comprehensive Strategy for Accurate Mutation Detection of the Highly Homologous PMS2.

PubMed

Li, Jianli; Dai, Hongzheng; Feng, Yanming; Tang, Jia; Chen, Stella; Tian, Xia; Gorman, Elizabeth; Schmitt, Eric S; Hansen, Terah A A; Wang, Jing; Plon, Sharon E; Zhang, Victor Wei; Wong, Lee-Jun C

2015-09-01

Germline mutations in the DNA mismatch repair gene PMS2 underlie the cancer susceptibility syndrome, Lynch syndrome. However, accurate molecular testing of PMS2 is complicated by a large number of highly homologous sequences. To establish a comprehensive approach for mutation detection of PMS2, we have designed a strategy combining targeted capture next-generation sequencing (NGS), multiplex ligation-dependent probe amplification, and long-range PCR followed by NGS to simultaneously detect point mutations and copy number changes of PMS2. Exonic deletions (E2 to E9, E5 to E9, E8, E10, E14, and E1 to E15), duplications (E11 to E12), and a nonsense mutation, p.S22*, were identified. Traditional multiplex ligation-dependent probe amplification and Sanger sequencing approaches cannot differentiate the origin of the exonic deletions in the 3' region when PMS2 and PMS2CL share identical sequences as a result of gene conversion. Our approach allows unambiguous identification of mutations in the active gene with a straightforward long-range-PCR/NGS method. Breakpoint analysis of multiple samples revealed that recurrent exon 14 deletions are mediated by homologous Alu sequences. Our comprehensive approach provides a reliable tool for accurate molecular analysis of genes containing multiple copies of highly homologous sequences and should improve PMS2 molecular analysis for patients with Lynch syndrome. Copyright © 2015 American Society for Investigative Pathology and the Association for Molecular Pathology. Published by Elsevier Inc. All rights reserved.

HLA DNA Sequence Variation among Human Populations: Molecular Signatures of Demographic and Selective Events

PubMed Central

Buhler, Stéphane; Sanchez-Mazas, Alicia

2011-01-01

Molecular differences between HLA alleles vary up to 57 nucleotides within the peptide binding coding region of human Major Histocompatibility Complex (MHC) genes, but it is still unclear whether this variation results from a stochastic process or from selective constraints related to functional differences among HLA molecules. Although HLA alleles are generally treated as equidistant molecular units in population genetic studies, DNA sequence diversity among populations is also crucial to interpret the observed HLA polymorphism. In this study, we used a large dataset of 2,062 DNA sequences defined for the different HLA alleles to analyze nucleotide diversity of seven HLA genes in 23,500 individuals of about 200 populations spread worldwide. We first analyzed the HLA molecular structure and diversity of these populations in relation to geographic variation and we further investigated possible departures from selective neutrality through Tajima's tests and mismatch distributions. All results were compared to those obtained by classical approaches applied to HLA allele frequencies. Our study shows that the global patterns of HLA nucleotide diversity among populations are significantly correlated to geography, although in some specific cases the molecular information reveals unexpected genetic relationships. At all loci except HLA-DPB1, populations have accumulated a high proportion of very divergent alleles, suggesting an advantage of heterozygotes expressing molecularly distant HLA molecules (asymmetric overdominant selection model). However, both different intensities of selection and unequal levels of gene conversion may explain the heterogeneous mismatch distributions observed among the loci. Also, distinctive patterns of sequence divergence observed at the HLA-DPB1 locus suggest current neutrality but old selective pressures on this gene. We conclude that HLA DNA sequences advantageously complement HLA allele frequencies as a source of data used to explore the genetic history of human populations, and that their analysis allows a more thorough investigation of human MHC molecular evolution. PMID:21408106

Complete mitochondrial genome sequences from five Eimeria species (Apicomplexa; Coccidia; Eimeriidae) infecting domestic turkeys

PubMed Central

2014-01-01

Background Clinical and subclinical coccidiosis is cosmopolitan and inflicts significant losses to the poultry industry globally. Seven named Eimeria species are responsible for coccidiosis in turkeys: Eimeria dispersa; Eimeria meleagrimitis; Eimeria gallopavonis; Eimeria meleagridis; Eimeria adenoeides; Eimeria innocua; and, Eimeria subrotunda. Although attempts have been made to characterize these parasites molecularly at the nuclear 18S rDNA and ITS loci, the maternally-derived and mitotically replicating mitochondrial genome may be more suited for species level molecular work; however, only limited sequence data are available for Eimeria spp. infecting turkeys. The purpose of this study was to sequence and annotate the complete mitochondrial genomes from 5 Eimeria species that commonly infect the domestic turkey (Meleagris gallopavo). Methods Six single-oocyst derived cultures of five Eimeria species infecting turkeys were PCR-amplified and sequenced completely prior to detailed annotation. Resulting sequences were aligned and used in phylogenetic analyses (BI, ML, and MP) that included complete mitochondrial genomes from 16 Eimeria species or concatenated CDS sequences from each genome. Results Complete mitochondrial genome sequences were obtained for Eimeria adenoeides Guelph, 6211 bp; Eimeria dispersa Briston, 6238 bp; Eimeria meleagridis USAR97-01, 6212 bp; Eimeria meleagrimitis USMN08-01, 6165 bp; Eimeria gallopavonis Weybridge, 6215 bp; and Eimeria gallopavonis USKS06-01, 6215 bp). The order, orientation and CDS lengths of the three protein coding genes (COI, COIII and CytB) as well as rDNA fragments encoding ribosomal large and small subunit rRNA were conserved among all sequences. Pairwise sequence identities between species ranged from 88.1% to 98.2%; sequence variability was concentrated within CDS or between rDNA fragments (where indels were common). No phylogenetic reconstruction supported monophyly of Eimeria species infecting turkeys; Eimeria dispersa may have arisen via host switching from another avian host. Phylogenetic analyses suggest E. necatrix and E. tenella are related distantly to other Eimeria of chickens. Conclusions Mitochondrial genomes of Eimeria species sequenced to date are highly conserved with regard to gene content and structure. Nonetheless, complete mitochondrial genome sequences and, particularly the three CDS, possess sufficient sequence variability for differentiating Eimeria species of poultry. The mitochondrial genome sequences are highly suited for molecular diagnostics and phylogenetics of coccidia and, potentially, genetic markers for molecular epidemiology. PMID:25034633

Complete mitochondrial genome sequences from five Eimeria species (Apicomplexa; Coccidia; Eimeriidae) infecting domestic turkeys.

PubMed

Ogedengbe, Mosun E; El-Sherry, Shiem; Whale, Julia; Barta, John R

2014-07-17

Clinical and subclinical coccidiosis is cosmopolitan and inflicts significant losses to the poultry industry globally. Seven named Eimeria species are responsible for coccidiosis in turkeys: Eimeria dispersa; Eimeria meleagrimitis; Eimeria gallopavonis; Eimeria meleagridis; Eimeria adenoeides; Eimeria innocua; and, Eimeria subrotunda. Although attempts have been made to characterize these parasites molecularly at the nuclear 18S rDNA and ITS loci, the maternally-derived and mitotically replicating mitochondrial genome may be more suited for species level molecular work; however, only limited sequence data are available for Eimeria spp. infecting turkeys. The purpose of this study was to sequence and annotate the complete mitochondrial genomes from 5 Eimeria species that commonly infect the domestic turkey (Meleagris gallopavo). Six single-oocyst derived cultures of five Eimeria species infecting turkeys were PCR-amplified and sequenced completely prior to detailed annotation. Resulting sequences were aligned and used in phylogenetic analyses (BI, ML, and MP) that included complete mitochondrial genomes from 16 Eimeria species or concatenated CDS sequences from each genome. Complete mitochondrial genome sequences were obtained for Eimeria adenoeides Guelph, 6211 bp; Eimeria dispersa Briston, 6238 bp; Eimeria meleagridis USAR97-01, 6212 bp; Eimeria meleagrimitis USMN08-01, 6165 bp; Eimeria gallopavonis Weybridge, 6215 bp; and Eimeria gallopavonis USKS06-01, 6215 bp). The order, orientation and CDS lengths of the three protein coding genes (COI, COIII and CytB) as well as rDNA fragments encoding ribosomal large and small subunit rRNA were conserved among all sequences. Pairwise sequence identities between species ranged from 88.1% to 98.2%; sequence variability was concentrated within CDS or between rDNA fragments (where indels were common). No phylogenetic reconstruction supported monophyly of Eimeria species infecting turkeys; Eimeria dispersa may have arisen via host switching from another avian host. Phylogenetic analyses suggest E. necatrix and E. tenella are related distantly to other Eimeria of chickens. Mitochondrial genomes of Eimeria species sequenced to date are highly conserved with regard to gene content and structure. Nonetheless, complete mitochondrial genome sequences and, particularly the three CDS, possess sufficient sequence variability for differentiating Eimeria species of poultry. The mitochondrial genome sequences are highly suited for molecular diagnostics and phylogenetics of coccidia and, potentially, genetic markers for molecular epidemiology.

In-Gel Determination of L-Amino Acid Oxidase Activity Based on the Visualization of Prussian Blue-Forming Reaction

PubMed Central

Zhou, Ning; Zhao, Chuntian

2013-01-01

L-amino acid oxidase (LAAO) is attracting increasing attention due to its important functions. Diverse detection methods with their own properties have been developed for characterization of LAAO. In the present study, a simple, rapid, sensitive, cost-effective and reproducible method for quantitative in-gel determination of LAAO activity based on the visualization of Prussian blue-forming reaction is described. Coupled with SDS-PAGE, this Prussian blue agar assay can be directly used to determine the numbers and approximate molecular weights of LAAO in one step, allowing straightforward application for purification and sequence identification of LAAO from diverse samples. PMID:23383337

[Molecular-biological properties of the rubella virus strains isolated in St. Petersburg].

PubMed

Buzitskaia, Zh V; Sirotkin, A K; Gudkova, T M; Prochukhanova, A P; Karpov, A V; Tsybalova, L M; Kiselev, O I

2012-01-01

In the surveillance of rubella in the northwest region of Russia samples of nasopharyngeal swabs from 37 patients with rubella, which were treated in the 442nd district military hospital named after Z.P. Solovyov in autumn 2007 were screened for the rubella virus using RK-13 cell line, 22 strains of rubella virus were isolated. Gene sequencing of E1 region of rubella virus isolates was carried out. Rubella virus strains isolated in St. Petersburg during the 2007 outbreak belonged to rubella virus genotype 1E. The morphogenesis of RK-13 cells with formation of replication complexes and enveloped virions of rubella virus was shown.

The RNA-induced silencing complex: a versatile gene-silencing machine.

PubMed

Pratt, Ashley J; MacRae, Ian J

2009-07-03

RNA interference is a powerful mechanism of gene silencing that underlies many aspects of eukaryotic biology. On the molecular level, RNA interference is mediated by a family of ribonucleoprotein complexes called RNA-induced silencing complexes (RISCs), which can be programmed to target virtually any nucleic acid sequence for silencing. The ability of RISC to locate target RNAs has been co-opted by evolution many times to generate a broad spectrum of gene-silencing pathways. Here, we review the fundamental biochemical and biophysical properties of RISC that facilitate gene targeting and describe the various mechanisms of gene silencing known to exploit RISC activity.

Haemophilus parainfluenzae meningitis in an adult associated with acute otitis media.

PubMed

Cardines, Rita; Giufrè, Maria; Ciofi degli Atti, Marta Luisa; Accogli, Marisa; Mastrantonio, Paola; Cerquetti, Marina

2009-04-01

A case of Haemophilus parainfluenzae meningitis in a woman with a 3-day history of acute otitis media is reported. Her medical history included bladder cancer surgery. Unequivocal identification of the isolate was obtained by using molecular techniques such as 16S rRNA sequencing besides to conventional culture methods. To investigate bacterial virulent traits possibly related to invasive properties, in vitro serum resistance of our isolate was analyzed, but it was found serum susceptible. Our study demonstrates that H. parainfluenzae can be considered an opportunistic pathogen able to cause life-threatening infections not only in children but also in patients with underlying conditions.

The Comprehensive Microbial Resource.

PubMed

Peterson, J D; Umayam, L A; Dickinson, T; Hickey, E K; White, O

2001-01-01

One challenge presented by large-scale genome sequencing efforts is effective display of uniform information to the scientific community. The Comprehensive Microbial Resource (CMR) contains robust annotation of all complete microbial genomes and allows for a wide variety of data retrievals. The bacterial information has been placed on the Web at http://www.tigr.org/CMR for retrieval using standard web browsing technology. Retrievals can be based on protein properties such as molecular weight or hydrophobicity, GC-content, functional role assignments and taxonomy. The CMR also has special web-based tools to allow data mining using pre-run homology searches, whole genome dot-plots, batch downloading and traversal across genomes using a variety of datatypes.

Computational Approaches for Revealing the Structure of Membrane Transporters: Case Study on Bilitranslocase.

PubMed

Venko, Katja; Roy Choudhury, A; Novič, Marjana

2017-01-01

The structural and functional details of transmembrane proteins are vastly underexplored, mostly due to experimental difficulties regarding their solubility and stability. Currently, the majority of transmembrane protein structures are still unknown and this present a huge experimental and computational challenge. Nowadays, thanks to X-ray crystallography or NMR spectroscopy over 3000 structures of membrane proteins have been solved, among them only a few hundred unique ones. Due to the vast biological and pharmaceutical interest in the elucidation of the structure and the functional mechanisms of transmembrane proteins, several computational methods have been developed to overcome the experimental gap. If combined with experimental data the computational information enables rapid, low cost and successful predictions of the molecular structure of unsolved proteins. The reliability of the predictions depends on the availability and accuracy of experimental data associated with structural information. In this review, the following methods are proposed for in silico structure elucidation: sequence-dependent predictions of transmembrane regions, predictions of transmembrane helix-helix interactions, helix arrangements in membrane models, and testing their stability with molecular dynamics simulations. We also demonstrate the usage of the computational methods listed above by proposing a model for the molecular structure of the transmembrane protein bilitranslocase. Bilitranslocase is bilirubin membrane transporter, which shares similar tissue distribution and functional properties with some of the members of the Organic Anion Transporter family and is the only member classified in the Bilirubin Transporter Family. Regarding its unique properties, bilitranslocase is a potentially interesting drug target.

Molecular Cloning and Pharmacological Properties of an Acidic PLA2 from Bothrops pauloensis Snake Venom

PubMed Central

Ferreira, Francis Barbosa; Gomes, Mário Sérgio Rocha; Naves de Souza, Dayane Lorena; Gimenes, Sarah Natalie Cirilo; Castanheira, Letícia Eulalio; Borges, Márcia Helena; Rodrigues, Renata Santos; Yoneyama, Kelly Aparecida Geraldo; Homsi Brandeburgo, Maria Inês; Rodrigues, Veridiana M.

2013-01-01

In this work, we describe the molecular cloning and pharmacological properties of an acidic phospholipase A2 (PLA2) isolated from Bothrops pauloensis snake venom. This enzyme, denominated BpPLA2-TXI, was purified by four chromatographic steps and represents 2.4% of the total snake venom protein content. BpPLA2-TXI is a monomeric protein with a molecular mass of 13.6 kDa, as demonstrated by Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF) analysis and its theoretical isoelectric point was 4.98. BpPLA2-TXI was catalytically active and showed some pharmacological effects such as inhibition of platelet aggregation induced by collagen or ADP and also induced edema and myotoxicity. BpPLA2-TXI displayed low cytotoxicity on TG-180 (CCRF S 180 II) and Ovarian Carcinoma (OVCAR-3), whereas no cytotoxicity was found in regard to MEF (Mouse Embryonic Fibroblast) and Sarcoma 180 (TIB-66). The N-terminal sequence of forty-eight amino acid residues was determined by Edman degradation. In addition, the complete primary structure of 122 amino acids was deduced by cDNA from the total RNA of the venom gland using specific primers, and it was significantly similar to other acidic D49 PLA2s. The phylogenetic analyses showed that BpPLA2-TXI forms a group with other acidic D49 PLA2s from the gender Bothrops, which are characterized by a catalytic activity associated with anti-platelet effects. PMID:24304676

A novel truncation mutation in CRYBB1 associated with autosomal dominant congenital cataract with nystagmus.

PubMed

Rao, Yan; Dong, Sufang; Li, Zuhua; Yang, Guohua; Peng, Chunyan; Yan, Ming; Zheng, Fang

2017-01-01

To identify the potential candidate genes for a large Chinese family with autosomal dominant congenital cataract (ADCC) and nystagmus, and investigate the possible molecular mechanism underlying the role of the candidate genes in cataractogenesis. We combined the linkage analysis and direct sequencing for the candidate genes in the linkage regions to identify the causative mutation. The molecular and bio-functional properties of the proteins encoded by the candidate genes was further explored with biophysical and biochemical studies of the recombinant wild-type and mutant proteins. We identified a c. C749T (p.Q227X) transversion in exon 6 of CRYBB1 , a cataract-causative gene. This nonsense mutation changes a phylogenetically conserved glutamine to a stop codon and is predicted to truncate the C-terminus of the wild-type protein by 26 amino acids. Comparison of the biophysical and biochemical properties of the recombinant full-length and truncated βB1-crystallins revealed that the mutation led to the insolubility and the phase separation phenomenon of the truncated protein with a changed conformation. Meanwhile, the thermal stability of the truncated βB1-crystallin was significantly decreased, and the mutation diminished the chaperoning ability of αA-crystallin with the mutant under heating stress. Our findings highlight the importance of the C-terminus in βB1-crystallin in maintaining the crystalline function and stability, and provide a novel insight into the molecular mechanism underlying the pathogenesis of human autosomal dominant congenital cataract.