Generate Optimized Genetic Rhythm for Enzyme Expression in Non-native systems

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

2016-11-03

Most amino acids are represented by more than one codon, resulting in redundancy in the genetic code. Silent codon substitutions that do not alter the amino acid sequence still have an effect on protein expression. We have developed an algorithm, GoGREEN, to enhance the expression of foreign proteins in a host organism. GoGREEN selects codons according to frequency patterns seen in the gene of interest using the codon usage table from the host organism. GoGREEN is also designed to accommodate gaps in the sequence.This software takes for input (1) the aligned protein sequences for genes the user wishes to express,more » (2) the codon usage table for the host organism, (3) and the DNA sequence for the target protein found in the host organism. The program will select codons based on codon usage patterns for the target DNA sequence. The program will also select codons for “gaps” found in the aligned protein sequences using the codon usage table from the host organism.« less

Emergent Rules for Codon Choice Elucidated by Editing Rare Arginine Codons in Escherichia coli

DTIC Science & Technology

2016-09-20

alternative codons are more likely to be viable. To evaluate synonymous and nonsynonymous alternatives to essential AGRs further, we imple- mented a CRISPR ... Crispr -assisted MAGE). First, we designed oligos that changed not only the target AGR codon to NNN but also made several synonymous changes at least 50...nt downstream that would disrupt a 20-bp CRISPR target lo- cus. MAGE was used to replace each AGR with NNN in parallel, and CRISPR /cas9 was used to

Succinyl-CoA:3-ketoacid CoA transferase (SCOT) deficiency: two pathogenic mutations, V133E and C456F, in Japanese siblings.

PubMed

Song, X Q; Fukao, T; Watanabe, H; Shintaku, H; Hirayama, K; Kassovska-Bratinova, S; Kondo, N; Mitchell, G A

1998-01-01

Succinyl-CoA:3-ketoacid CoA transferase (SCOT; EC 2.8.3.5; locus symbol OXCT) is the key enzyme of ketone body utilization. Hereditary SCOT deficiency (MIM 245050) causes episodes of severe ketoacidosis. We developed a transient expression system for mutant SCOT cDNAs, using immortalized SCOT-deficient fibroblasts. This paper describes and characterizes three missense mutations in two SCOT-deficient siblings from Japan. They are genetic compounds who inherited the mutation C456F (c1367 G-->T) from their mother. Their paternal allele contains two mutations in cis, T58M (c173 C-->T) and V133E (c398T-->A). Expression of SCOT cDNAs containing either V133E or C456F produces no detectable SCOT activity, whereas T58M is functionally neutral. T58M is a rare sequence variant not detected in 100 control Japanese alleles. In fibroblasts from the proband (GS02), in whom immunoblot demonstrated no detectable SCOT peptide, we measured an apparent residual SCOT activity of 20-35%. We hypothesize that the high residual SCOT activity in homogenates may be an artifact caused by use of the substrate, acetoacetyl-CoA by other enzymes. Expression of mutant SCOT cDNAs more accurately reflects the residual activity of SCOT than do currently available assays in cell or tissue homogenates.

Better Incident Response with SCOT

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

Bruner, Todd

2015-04-01

SCOT is an incident response management system and knowledge base designed for incident responders by incident responders. SCOT increases the effectiveness of the team without adding undue burdens. Focused on reducing the friction between analysts and their tools, SCOT enables analysts to document and share their research and response efforts in near real time. Automatically identifying indicators and correlating those indicators, SCOT helps analysts discover and respond to advanced threats.

Effects of the Huguenot Diaspora on the American Revolution

DTIC Science & Technology

2016-06-10

99 Scots from Scotland and other warmer Locales...106 viii The Scots -Irish or Ulster Scots : Huguenots through the Back Door .......................... 111 The Minor Streams...immigrated to the Shenandoah Valley by way of Baltimore, Maryland. Over a couple of generations the family assimilated into the Scots

Analysis of Serine Codon Conservation Reveals Diverse Phenotypic Constraints on Hepatitis C Virus Glycoprotein Evolution

PubMed Central

Koutsoudakis, George; Urbanowicz, Richard A.; Mirza, Deeman; Ginkel, Corinne; Riebesehl, Nina; Calland, Noémie; Albecka, Anna; Price, Louisa; Hudson, Natalia; Descamps, Véronique; Backx, Matthijs; McClure, C. Patrick; Duverlie, Gilles; Pecheur, Eve-Isabelle; Dubuisson, Jean; Perez-del-Pulgar, Sofia; Forns, Xavier; Steinmann, Eike; Tarr, Alexander W.; Pietschmann, Thomas

2014-01-01

Serine is encoded by two divergent codon types, UCN and AGY, which are not interchangeable by a single nucleotide substitution. Switching between codon types therefore occurs via intermediates (threonine or cysteine) or via simultaneous tandem substitutions. Hepatitis C virus (HCV) chronically infects 2 to 3% of the global population. The highly variable glycoproteins E1 and E2 decorate the surface of the viral envelope, facilitate cellular entry, and are targets for host immunity. Comparative sequence analysis of globally sampled E1E2 genes, coupled with phylogenetic analysis, reveals the signatures of multiple archaic codon-switching events at seven highly conserved serine residues. Limited detection of intermediate phenotypes indicates that associated fitness costs restrict their fixation in divergent HCV lineages. Mutational pathways underlying codon switching were probed via reverse genetics, assessing glycoprotein functionality using multiple in vitro systems. These data demonstrate selection against intermediate phenotypes can act at the structural/functional level, with some intermediates displaying impaired virion assembly and/or decreased capacity for target cell entry. These effects act in residue/isolate-specific manner. Selection against intermediates is also provided by humoral targeting, with some intermediates exhibiting increased epitope exposure and enhanced neutralization sensitivity, despite maintaining a capacity for target cell entry. Thus, purifying selection against intermediates limits their frequencies in globally sampled strains, with divergent functional constraints at the protein level restricting the fixation of deleterious mutations. Overall our study provides an experimental framework for identification of barriers limiting viral substitutional evolution and indicates that serine codon-switching represents a genomic “fossil record” of historical purifying selection against E1E2 intermediate phenotypes. PMID:24173227

CRISPR-Mediated Base Editing Enables Efficient Disruption of Eukaryotic Genes through Induction of STOP Codons.

PubMed

Billon, Pierre; Bryant, Eric E; Joseph, Sarah A; Nambiar, Tarun S; Hayward, Samuel B; Rothstein, Rodney; Ciccia, Alberto

2017-09-21

Standard CRISPR-mediated gene disruption strategies rely on Cas9-induced DNA double-strand breaks (DSBs). Here, we show that CRISPR-dependent base editing efficiently inactivates genes by precisely converting four codons (CAA, CAG, CGA, and TGG) into STOP codons without DSB formation. To facilitate gene inactivation by induction of STOP codons (iSTOP), we provide access to a database of over 3.4 million single guide RNAs (sgRNAs) for iSTOP (sgSTOPs) targeting 97%-99% of genes in eight eukaryotic species, and we describe a restriction fragment length polymorphism (RFLP) assay that allows the rapid detection of iSTOP-mediated editing in cell populations and clones. To simplify the selection of sgSTOPs, our resource includes annotations for off-target propensity, percentage of isoforms targeted, prediction of nonsense-mediated decay, and restriction enzymes for RFLP analysis. Additionally, our database includes sgSTOPs that could be employed to precisely model over 32,000 cancer-associated nonsense mutations. Altogether, this work provides a comprehensive resource for DSB-free gene disruption by iSTOP. Copyright © 2017 Elsevier Inc. All rights reserved.

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

PubMed Central

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

2016-01-01

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

Automated design of degenerate codon libraries.

PubMed

Mena, Marco A; Daugherty, Patrick S

2005-12-01

Degenerate codon libraries are frequently used in protein engineering and evolution studies but are often limited to targeting a small number of positions to adequately limit the search space. To mitigate this, codon degeneracy can be limited using heuristics or previous knowledge of the targeted positions. To automate design of libraries given a set of amino acid sequences, an algorithm (LibDesign) was developed that generates a set of possible degenerate codon libraries, their resulting size, and their score relative to a user-defined scoring function. A gene library of a specified size can then be constructed that is representative of the given amino acid distribution or that includes specific sequences or combinations thereof. LibDesign provides a new tool for automated design of high-quality protein libraries that more effectively harness existing sequence-structure information derived from multiple sequence alignment or computational protein design data.

A Prelude to Revolution: Scots-Irish Vigilantes in the Colonial Backcountry

DTIC Science & Technology

2014-06-13

A PRELUDE TO REVOLUTION: SCOTS -IRISH VIGILANTES IN THE COLONIAL BACKCOUNTRY A thesis presented to the Faculty of the U.S...2014 4. TITLE AND SUBTITLE A Prelude to Revolution: Scots -Irish Vigilantes in the Colonial Backcountry 5a. CONTRACT NUMBER 5b. GRANT...arrived Scots -Irish. These clannish warrior farmers from the north of Ireland ultimately broke out in open rebellion in several locations in the

Ribosomes slide on lysine-encoding homopolymeric A stretches

PubMed Central

Koutmou, Kristin S; Schuller, Anthony P; Brunelle, Julie L; Radhakrishnan, Aditya; Djuranovic, Sergej; Green, Rachel

2015-01-01

Protein output from synonymous codons is thought to be equivalent if appropriate tRNAs are sufficiently abundant. Here we show that mRNAs encoding iterated lysine codons, AAA or AAG, differentially impact protein synthesis: insertion of iterated AAA codons into an ORF diminishes protein expression more than insertion of synonymous AAG codons. Kinetic studies in E. coli reveal that differential protein production results from pausing on consecutive AAA-lysines followed by ribosome sliding on homopolymeric A sequence. Translation in a cell-free expression system demonstrates that diminished output from AAA-codon-containing reporters results from premature translation termination on out of frame stop codons following ribosome sliding. In eukaryotes, these premature termination events target the mRNAs for Nonsense-Mediated-Decay (NMD). The finding that ribosomes slide on homopolymeric A sequences explains bioinformatic analyses indicating that consecutive AAA codons are under-represented in gene-coding sequences. Ribosome ‘sliding’ represents an unexpected type of ribosome movement possible during translation. DOI: http://dx.doi.org/10.7554/eLife.05534.001 PMID:25695637

Functional Versatility of AGY Serine Codons in Immunoglobulin Variable Region Genes

PubMed Central

Detanico, Thiago; Phillips, Matthew; Wysocki, Lawrence J.

2016-01-01

In systemic autoimmunity, autoantibodies directed against nuclear antigens (Ags) often arise by somatic hypermutation (SHM) that converts AGT and AGC (AGY) Ser codons into Arg codons. This can occur by three different single-base changes. Curiously, AGY Ser codons are far more abundant in complementarity-determining regions (CDRs) of IgV-region genes than expected for random codon use or from species-specific codon frequency data. CDR AGY codons are also more abundant than TCN Ser codons. We show that these trends hold even in cartilaginous fishes. Because AGC is a preferred target for SHM by activation-induced cytidine deaminase, we asked whether the AGY abundance was solely due to a selection pressure to conserve high mutability in CDRs regardless of codon context but found that this was not the case. Instead, AGY triplets were selectively enriched in the Ser codon reading frame. Motivated by reports implicating a functional role for poly/autoreactive specificities in antiviral antibodies, we also analyzed mutations at AGY in antibodies directed against a number of different viruses and found that mutations producing Arg codons in antiviral antibodies were indeed frequent. Unexpectedly, however, we also found that AGY codons mutated often to encode nearly all of the amino acids that are reported to provide the most frequent contacts with Ag. In many cases, mutations producing codons for these alternative amino acids in antiviral antibodies were more frequent than those producing Arg codons. Mutations producing each of these key amino acids required only single-base changes in AGY. AGY is the only codon group in which two-thirds of random mutations generate codons for these key residues. Finally, by directly analyzing X-ray structures of immune complexes from the RCSB protein database, we found that Ag-contact residues generated via SHM occurred more often at AGY than at any other codon group. Thus, preservation of AGY codons in antibody genes appears to have been driven by their exceptional functional versatility, despite potential autoreactive consequences. PMID:27920779

Modulation of c-fms proto-oncogene in an ovarian carcinoma cell line by a hammerhead ribozyme.

PubMed Central

Yokoyama, Y.; Morishita, S.; Takahashi, Y.; Hashimoto, M.; Tamaya, T.

1997-01-01

Co-expression of macrophage colony-stimulating factor (M-CSF) and its receptor (c-fms) is often found in ovarian epithelial carcinoma, suggesting the existence of autocrine regulation of cell growth by M-CSF. To block this autocrine loop, we have developed hammerhead ribozymes against c-fms mRNA. As target sites of the ribozyme, we chose the GUC sequence in codon 18 and codon 27 of c-fms mRNA. Two kinds of ribozymes were able to cleave an artificial c-fms RNA substrate in a cell-free system, although the ribozyme against codon 18 was much more efficient than that against codon 27. We next constructed an expression vector carrying a ribozyme sequence that targeted the GUC sequence in codon 18 of c-fms mRNA. It was introduced into TYK-nu cells that expressed M-CSF and its receptor. Its transfectant showed a reduced growth potential. The expression levels of c-fms protein and mRNA in the transfectant were clearly decreased with the expression of ribozyme RNA compared with that of an untransfected control or a transfectant with the vector without the ribozyme sequence. These results suggest that the ribozyme against GUC in codon 18 of c-fms mRNA is a promising tool for blocking the autocrine loop of M-CSF in ovarian epithelial carcinoma. Images Figure 2 Figure 3 Figure 5 Figure 6 PMID:9376277

Sequence similarity is more relevant than species specificity in probabilistic backtranslation.

PubMed

Ferro, Alfredo; Giugno, Rosalba; Pigola, Giuseppe; Pulvirenti, Alfredo; Di Pietro, Cinzia; Purrello, Michele; Ragusa, Marco

2007-02-21

Backtranslation is the process of decoding a sequence of amino acids into the corresponding codons. All synthetic gene design systems include a backtranslation module. The degeneracy of the genetic code makes backtranslation potentially ambiguous since most amino acids are encoded by multiple codons. The common approach to overcome this difficulty is based on imitation of codon usage within the target species. This paper describes EasyBack, a new parameter-free, fully-automated software for backtranslation using Hidden Markov Models. EasyBack is not based on imitation of codon usage within the target species, but instead uses a sequence-similarity criterion. The model is trained with a set of proteins with known cDNA coding sequences, constructed from the input protein by querying the NCBI databases with BLAST. Unlike existing software, the proposed method allows the quality of prediction to be estimated. When tested on a group of proteins that show different degrees of sequence conservation, EasyBack outperforms other published methods in terms of precision. The prediction quality of a protein backtranslation methis markedly increased by replacing the criterion of most used codon in the same species with a Hidden Markov Model trained with a set of most similar sequences from all species. Moreover, the proposed method allows the quality of prediction to be estimated probabilistically.

Confirmation of translatability and functionality certifies the dual endothelin1/VEGFsp receptor (DEspR) protein.

PubMed

Herrera, Victoria L M; Steffen, Martin; Moran, Ann Marie; Tan, Glaiza A; Pasion, Khristine A; Rivera, Keith; Pappin, Darryl J; Ruiz-Opazo, Nelson

2016-06-14

In contrast to rat and mouse databases, the NCBI gene database lists the human dual-endothelin1/VEGFsp receptor (DEspR, formerly Dear) as a unitary transcribed pseudogene due to a stop [TGA]-codon at codon#14 in automated DNA and RNA sequences. However, re-analysis is needed given prior single gene studies detected a tryptophan [TGG]-codon#14 by manual Sanger sequencing, demonstrated DEspR translatability and functionality, and since the demonstration of actual non-translatability through expression studies, the standard-of-excellence for pseudogene designation, has not been performed. Re-analysis must meet UNIPROT criteria for demonstration of a protein's existence at the highest (protein) level, which a priori, would override DNA- or RNA-based deductions. To dissect the nucleotide sequence discrepancy, we performed Maxam-Gilbert sequencing and reviewed 727 RNA-seq entries. To comply with the highest level multiple UNIPROT criteria for determining DEspR's existence, we performed various experiments using multiple anti-DEspR monoclonal antibodies (mAbs) targeting distinct DEspR epitopes with one spanning the contested tryptophan [TGG]-codon#14, assessing: (a) DEspR protein expression, (b) predicted full-length protein size, (c) sequence-predicted protein-specific properties beyond codon#14: receptor glycosylation and internalization, (d) protein-partner interactions, and (e) DEspR functionality via DEspR-inhibition effects. Maxam-Gilbert sequencing and some RNA-seq entries demonstrate two guanines, hence a tryptophan [TGG]-codon#14 within a compression site spanning an error-prone compression sequence motif. Western blot analysis using anti-DEspR mAbs targeting distinct DEspR epitopes detect the identical glycosylated 17.5 kDa pull-down protein. Decrease in DEspR-protein size after PNGase-F digest demonstrates post-translational glycosylation, concordant with the consensus-glycosylation site beyond codon#14. Like other small single-transmembrane proteins, mass spectrometry analysis of anti-DEspR mAb pull-down proteins do not detect DEspR, but detect DEspR-protein interactions with proteins implicated in intracellular trafficking and cancer. FACS analyses also detect DEspR-protein in different human cancer stem-like cells (CSCs). DEspR-inhibition studies identify DEspR-roles in CSC survival and growth. Live cell imaging detects fluorescently-labeled anti-DEspR mAb targeted-receptor internalization, concordant with the single internalization-recognition sequence also located beyond codon#14. Data confirm translatability of DEspR, the full-length DEspR protein beyond codon#14, and elucidate DEspR-specific functionality. Along with detection of the tryptophan [TGG]-codon#14 within an error-prone compression site, cumulative data demonstrating DEspR protein existence fulfill multiple UNIPROT criteria, thus refuting its pseudogene designation.

Codon Optimization to Enhance Expression Yields Insights into Chloroplast Translation1[OPEN

PubMed Central

Chan, Hui-Ting; Williams-Carrier, Rosalind; Barkan, Alice

2016-01-01

Codon optimization based on psbA genes from 133 plant species eliminated 105 (human clotting factor VIII heavy chain [FVIII HC]) and 59 (polio VIRAL CAPSID PROTEIN1 [VP1]) rare codons; replacement with only the most highly preferred codons decreased transgene expression (77- to 111-fold) when compared with the codon usage hierarchy of the psbA genes. Targeted proteomic quantification by parallel reaction monitoring analysis showed 4.9- to 7.1-fold or 22.5- to 28.1-fold increase in FVIII or VP1 codon-optimized genes when normalized with stable isotope-labeled standard peptides (or housekeeping protein peptides), but quantitation using western blots showed 6.3- to 8-fold or 91- to 125-fold increase of transgene expression from the same batch of materials, due to limitations in quantitative protein transfer, denaturation, solubility, or stability. Parallel reaction monitoring, to our knowledge validated here for the first time for in planta quantitation of biopharmaceuticals, is especially useful for insoluble or multimeric proteins required for oral drug delivery. Northern blots confirmed that the increase of codon-optimized protein synthesis is at the translational level rather than any impact on transcript abundance. Ribosome footprints did not increase proportionately with VP1 translation or even decreased after FVIII codon optimization but is useful in diagnosing additional rate-limiting steps. A major ribosome pause at CTC leucine codons in the native gene of FVIII HC was eliminated upon codon optimization. Ribosome stalls observed at clusters of serine codons in the codon-optimized VP1 gene provide an opportunity for further optimization. In addition to increasing our understanding of chloroplast translation, these new tools should help to advance this concept toward human clinical studies. PMID:27465114

Aspheric and freeform surfaces metrology with software configurable optical test system: a computerized reverse Hartmann test

NASA Astrophysics Data System (ADS)

Su, Peng; Khreishi, Manal A. H.; Su, Tianquan; Huang, Run; Dominguez, Margaret Z.; Maldonado, Alejandro; Butel, Guillaume; Wang, Yuhao; Parks, Robert E.; Burge, James H.

2014-03-01

A software configurable optical test system (SCOTS) based on deflectometry was developed at the University of Arizona for rapidly, robustly, and accurately measuring precision aspheric and freeform surfaces. SCOTS uses a camera with an external stop to realize a Hartmann test in reverse. With the external camera stop as the reference, a coordinate measuring machine can be used to calibrate the SCOTS test geometry to a high accuracy. Systematic errors from the camera are carefully investigated and controlled. Camera pupil imaging aberration is removed with the external aperture stop. Imaging aberration and other inherent errors are suppressed with an N-rotation test. The performance of the SCOTS test is demonstrated with the measurement results from a 5-m-diameter Large Synoptic Survey Telescope tertiary mirror and an 8.4-m diameter Giant Magellan Telescope primary mirror. The results show that SCOTS can be used as a large-dynamic-range, high-precision, and non-null test method for precision aspheric and freeform surfaces. The SCOTS test can achieve measurement accuracy comparable to traditional interferometric tests.

Are Scots pine forest edges particularly prone to drought-stress?

NASA Astrophysics Data System (ADS)

Buras, Allan; Schunk, Christian; Taeger, Steffen; Lemme, Hannes; Gößwein, Sebastian; Menzel, Annette

2017-04-01

In 2016, Scots pine (Pinus sylvestris L.) forests experienced a pronounced dieback in several regions across Germany. Being an economically important tree species, a thorough identification of the reasons for this dieback is of high interest. The dieback is likely to be associated with a record drought event which occurred in summer 2015. However, visual observations indicate that forest edges were particularly affected. This observation is supported by a study from Sweden which showed that Scots pine trees growing at a north-facing forest edge expressed a higher water use if compared to trees from the interior (Cienciala et al., 2002). We therefore hypothesize that Scots pine trees are more prone to drought-stress induced dieback when growing at the forest edge. To test this hypothesis, we investigated the growth performance of Scots pine across three affected stands in Franconia, southern Germany. The stands were selected to represent differing conditions along a gradient of forest fragmentation, ranging from the forest interior, over a forest edge situation, to a small forest island. By means of dendroclimatology and UAV-borne remote sensing, Scots pine growth performance and vitality was compared among the three stands. Our results revealed differing Scots pine growth reactions between the forest interior and forest edge as indicated by the identification of different responder groups (Buras et al., 2016). The forest edge and the forest island expressed significantly higher correlations with the drought-index SPEI (Vicente-Serrano et al., 2009) if compared to the forest interior. Moreover, NDVI of Scots Pine canopies significantly decreased towards the forest edge, this indicating lower vitality of corresponding trees. In conclusion, our results highlight Scots pine to be more prone to drought-stress when growing at the forest edge. This finding has important implications for forest management activities in the context of climate change adaptation, since foresters may need to revise concepts of Scots pine management at forest edges and in forest islands under an increasingly warmer and drier climate. 1. Cienciala, E. et al. The effect of a north-facing forest edge on tree water use in a boreal Scots pine stand. Can. J. For. Res. 32, 693-702 (2002). 2. Buras, A. et al. Tuning the Voices of a Choir: Detecting Ecological Gradients in Time-Series Populations. PLOS ONE 11, e0158346 (2016). 3. Vicente-Serrano, S. M., Beguería, S. & López-Moreno, J. I. A Multiscalar Drought Index Sensitive to Global Warming: The Standardized Precipitation Evapotranspiration Index. J. Climate 23, 1696-1718 (2009).

Response of Scots pine stand vitality to changes in environmental factors in Poland, 1991-1995

Treesearch

Jerzy Wawrzoniak

1998-01-01

Vitality inventories of Scots pine stands, the most common species in Poland, have been done since 1991 by using the ICP-Forest methodology. In Scots pine stands older than 40 years, 1,040 observation plots were established. Defoliation was used as the primary indicator of stand vitality. During 1991 to 1995, SO2 and NOx...

Codon Optimization of the Human Papillomavirus E7 Oncogene Induces a CD8+ T Cell Response to a Cryptic Epitope Not Harbored by Wild-Type E7

PubMed Central

Lorenz, Felix K. M.; Wilde, Susanne; Voigt, Katrin; Kieback, Elisa; Mosetter, Barbara; Schendel, Dolores J.; Uckert, Wolfgang

2015-01-01

Codon optimization of nucleotide sequences is a widely used method to achieve high levels of transgene expression for basic and clinical research. Until now, immunological side effects have not been described. To trigger T cell responses against human papillomavirus, we incubated T cells with dendritic cells that were pulsed with RNA encoding the codon-optimized E7 oncogene. All T cell receptors isolated from responding T cell clones recognized target cells expressing the codon-optimized E7 gene but not the wild type E7 sequence. Epitope mapping revealed recognition of a cryptic epitope from the +3 alternative reading frame of codon-optimized E7, which is not encoded by the wild type E7 sequence. The introduction of a stop codon into the +3 alternative reading frame protected the transgene product from recognition by T cell receptor gene-modified T cells. This is the first experimental study demonstrating that codon optimization can render a transgene artificially immunogenic through generation of a dominant cryptic epitope. This finding may be of great importance for the clinical field of gene therapy to avoid rejection of gene-corrected cells and for the design of DNA- and RNA-based vaccines, where codon optimization may artificially add a strong immunogenic component to the vaccine. PMID:25799237

Unmanned Tactical Autonomous Control and Collaboration Coactive Design

DTIC Science & Technology

2016-06-01

AUTONOMOUS CONTROL AND COLLABORATION COACTIVE DESIGN by Matthew S. Zach June 2016 Thesis Advisor: Dan Boger Second Reader: Scot Miller...POSTGRADUATE SCHOOL June 2016 Approved by: Dan Boger, Ph.D. Thesis Advisor Scot Miller Second Reader Dan Boger, Ph.D...assistance, and mentoring, I remain most grateful. I begin by thanking Dr. Dan Boger and Scot Miller for taking me on as a thesis student. You

Unmanned Tactical Autonomous Control and Collaboration (UTACC) Campaign of Experimentation

DTIC Science & Technology

2016-09-01

Boger Second Reader: Scot Miller THIS PAGE INTENTIONALLY LEFT BLANK i REPORT DOCUMENTATION PAGE Form Approved OMB No. 0704-0188 Public...from the NAVAL POSTGRADUATE SCHOOL September 2016 Approved by: Dan Boger, Ph.D. Thesis Advisor Scot Miller Second Reader...Erik Keim forms the bedrock of this thesis. Next, I would like to thank Dr. Dan Boger and Scot Miller for taking me on as a thesis candidate and

Unmanned Tactical Autonomous Control and Collaboration Measures of Performance and Measures of Effectiveness

DTIC Science & Technology

2016-09-01

September 2016 Thesis Advisor: Dan Boger Co-Advisor: Scot Miller THIS PAGE INTENTIONALLY LEFT BLANK i REPORT DOCUMENTATION PAGE Form Approved... Scot Miller Co-Advisor Dan Boger Chair, Information Sciences Department iv THIS PAGE INTENTIONALLY LEFT BLANK v ABSTRACT As the technological...The authors would like to thank their advisors, Scot Miller and Dr. Dan Boger, for their incredibly helpful guidance and feedback on the development

Differential response of Scots pine seedlings to variable intensity and ratio of red and far-red light.

PubMed

Razzak, Abdur; Ranade, Sonali Sachin; Strand, Åsa; García-Gil, M R

2017-08-01

We investigated the response to increasing intensity of red (R) and far-R (FR) light and to a decrease in R:FR ratio in Pinus sylvestris L. (Scots pine) seedling. The results showed that FR high-irradiance response for hypocotyl elongation may be present in Scots pine and that this response is enhanced by increasing light intensity. However, both hypocotyl inhibition and pigment accumulation were more strongly affected by the R light compared with FR light. This is in contrast to previous reports in Arabidopsis thaliana (L.) Heynh. In the angiosperm, A. thaliana R light shows an overall milder effect on inhibition of hypocotyl elongation and on pigment biosynthesis compared with FR suggesting conifers and angiosperms respond very differently to the different light regimes. Scots pine shade avoidance syndrome with longer hypocotyls, shorter cotyledons and lower chlorophyll content in response to shade conditions resembles the response observed in A. thaliana. However, anthocyanin accumulation increased with shade in Scots pine, which again differs from what is known in angiosperms. Overall, the response of seedling development and physiology to R and FR light in Scots pine indicates that the regulatory mechanism for light response may differ between gymnosperms and angiosperms. © 2017 John Wiley & Sons Ltd.

Molecular evolution of the enzymes involved in the sphingolipid metabolism of Leishmania: selection pressure in relation to functional divergence and conservation.

PubMed

Mandlik, Vineetha; Shinde, Sonali; Singh, Shailza

2014-06-21

Selection pressure governs the relative mutability and the conservedness of a protein across the protein family. Biomolecules (DNA, RNA and proteins) continuously evolve under the effect of evolutionary pressure that arises as a consequence of the host parasite interaction. IPCS (Inositol phosphorylceramide synthase), SPL (Sphingosine-1-P lyase) and SPT (Serine palmitoyl transferase) represent three important enzymes involved in the sphingolipid metabolism of Leishmania. These enzymes are responsible for maintaining the viability and infectivity of the parasite and have been classified as druggable targets in the parasite metabolome. The present work relates to the role of selection pressure deciding functional conservedness and divergence of the drug targets. IPCS and SPL protein families appear to diverge from the SPT family. The three protein families were largely under the influence of purifying selection and were moderately conserved baring two residues in the IPCS protein which were under the influence of positive selection. To further explore the selection pressure at the codon level, codon usage bias indices were calculated to analyze genes for their synonymous codon usage pattern. IPCS gene exhibited slightly lower codon bias as compared to SPL and SPT protein families. Evolutionary tracing of the proposed drug targets has been done with a viewpoint that the amino-acids lining the drug binding pocket should have a lower evolvability. Sites under positive selection (HIS20 and CYS30 of IPCS) should be avoided during devising strategies for inhibitor design.

Above-Ground Dimensions and Acclimation Explain Variation in Drought Mortality of Scots Pine Seedlings from Various Provenances

PubMed Central

Seidel, Hannes; Menzel, Annette

2016-01-01

Seedling establishment is a critical part of the life cycle, thus seedling survival might be even more important for forest persistence under recent and future climate change. Scots pine forests have been disproportionally more affected by climate change triggered forest-dieback. Nevertheless, some Scots pine provenances might prove resilient to future drought events because of the species’ large distributional range, genetic diversity, and adaptation potential. However, there is a lack of knowledge on provenance-specific survival under severe drought events and on how acclimation alters survival rates in Scots pine seedlings. We therefore conducted two drought-induced mortality experiments with potted Scots pine seedlings in a greenhouse. In the first experiment, 760 three-year-old seedlings from 12 different provenances of the south-western distribution range were subjected to the same treatment followed by the mortality experiment in 2014. In the second experiment, we addressed the question of whether acclimation to re-occurring drought stress events and to elevated temperature might decrease mortality rates. Thus, 139 four-year-old seedlings from France, Germany, and Poland were subjected to different temperature regimes (2012–2014) and drought treatments (2013–2014) before the mortality experiment in 2015. Provenances clearly differed in their hazard of drought-induced mortality, which was only partly related to the climate of their origin. Drought acclimation decreased the hazard of drought-induced mortality. Above-ground dry weight and height were the main determinants for the hazard of mortality, i.e., heavier and taller seedlings were more prone to mortality. Consequently, Scots pine seedlings exhibit a considerable provenance-specific acclimation potential against drought mortality and the selection of suitable provenances might thus facilitate seedling establishment and the persistence of Scots pine forest. PMID:27458477

Drought-induced adaptation of the xylem in Scots pine and pubescent oak.

PubMed

Eilmann, Britta; Zweifel, Roman; Buchmann, Nina; Fonti, Patrick; Rigling, Andreas

2009-08-01

Drought impairs tree growth in the inner-Alpine valleys of Central Europe. We investigated species-specific responses to contrasting water supply, with Scots pine (Pinus sylvestris L.), threatened by drought-induced mortality, and pubescent oak (Quercus pubescens Willd.), showing no connection between drought events and mortality. The two co-occurring tree species were compared, growing either along an open water channel or at a site with naturally dry conditions. In addition, the growth response of Scots pine to a draining of a water channel was studied. We analysed the radial increment for the last 100 years and wood anatomical parameters for the last 45 years. Drought reduced the conduit area of pubescent oak, but increased the radial lumen diameter of the conduits in Scots pine. Both species decreased their radial increment under drought. In Scots pine, radial increment was generally more dependent on water availability than that in pubescent oak. Irrigated trees responded less negatively to high temperature as seen in the increase in the conduit area in pubescent oak and the removal of the limitation of cell division by high temperatures. After irrigation stopped, tree-ring width for Scots pine decreased within 1-year delay, whereas lumen diameter and cell-wall thickness responded with a 4-year delay. Scots pine seemed to optimize the carbon-per-conduit-costs under drought by increasing conduits diameter while decreasing cell numbers. This strategy might lead to a complete loss of tree rings under severe drought and thus to an impairment of water transport. In contrast, in pubescent oak tree-ring width is less affected by summer drought because parts of the earlywood are built in early spring. Thus, pubescent oak might have gradual advantages over pine in today's climate of the inner-Alpine valley.

Recovery plan for Scots pine blister rust caused by Cronartium flaccidum (Alb. & Schwein.) G. Winter and Peridermium pini (Pers.) Lév. [syn. C. asclepiadeum (Willd.) Fr., Endocronartium pini (Pers.) Y. Hiratsuka

Treesearch

Brian W. Geils; Ned B. Klopfenstein; Mee-Sook Kim; Pauline Spaine; Bryce A. Richardson; Paul J. Zambino; Charles G. Shaw; James Walla; Russ Bulluck; Laura Redmond; Kent Smith

2009-01-01

The sexually reproducing form of Scots pine blister rust, C. flaccidum, completes its life cycle alternating between pines of the subgenus Pinus and seed-plants of various families. Scots pine blister rust is also caused by a form of the rust that spreads directly from pine to pine and is named, Peridermium pini...

Improving End-To-End Tsunami Warning for Risk Reduction on Canada’s West Coast

DTIC Science & Technology

2015-01-01

Association Jason Wood , B.C. Ministry of Transportation and Infrastructure Steve Waugh, Bela Coola Dave Rainnie, Canadian Broadcasting Corporation...southern tip of Banks Island to the northern tip of Vancouver Island (the western limit of the eastern boundary of Cape Scot Provincial Park.) This... Scot to Port Renfrew Beginning on the eastern boundary of Cape Scot Provincial Park on the northern tip of Vancouver Island and running in a south

SCoT: a Python toolbox for EEG source connectivity.

PubMed

Billinger, Martin; Brunner, Clemens; Müller-Putz, Gernot R

2014-01-01

Analysis of brain connectivity has become an important research tool in neuroscience. Connectivity can be estimated between cortical sources reconstructed from the electroencephalogram (EEG). Such analysis often relies on trial averaging to obtain reliable results. However, some applications such as brain-computer interfaces (BCIs) require single-trial estimation methods. In this paper, we present SCoT-a source connectivity toolbox for Python. This toolbox implements routines for blind source decomposition and connectivity estimation with the MVARICA approach. Additionally, a novel extension called CSPVARICA is available for labeled data. SCoT estimates connectivity from various spectral measures relying on vector autoregressive (VAR) models. Optionally, these VAR models can be regularized to facilitate ill posed applications such as single-trial fitting. We demonstrate basic usage of SCoT on motor imagery (MI) data. Furthermore, we show simulation results of utilizing SCoT for feature extraction in a BCI application. These results indicate that CSPVARICA and correct regularization can significantly improve MI classification. While SCoT was mainly designed for application in BCIs, it contains useful tools for other areas of neuroscience. SCoT is a software package that (1) brings combined source decomposition and connectivtiy estimation to the open Python platform, and (2) offers tools for single-trial connectivity estimation. The source code is released under the MIT license and is available online at github.com/SCoT-dev/SCoT.

SCoT: a Python toolbox for EEG source connectivity

PubMed Central

Billinger, Martin; Brunner, Clemens; Müller-Putz, Gernot R.

2014-01-01

Analysis of brain connectivity has become an important research tool in neuroscience. Connectivity can be estimated between cortical sources reconstructed from the electroencephalogram (EEG). Such analysis often relies on trial averaging to obtain reliable results. However, some applications such as brain-computer interfaces (BCIs) require single-trial estimation methods. In this paper, we present SCoT—a source connectivity toolbox for Python. This toolbox implements routines for blind source decomposition and connectivity estimation with the MVARICA approach. Additionally, a novel extension called CSPVARICA is available for labeled data. SCoT estimates connectivity from various spectral measures relying on vector autoregressive (VAR) models. Optionally, these VAR models can be regularized to facilitate ill posed applications such as single-trial fitting. We demonstrate basic usage of SCoT on motor imagery (MI) data. Furthermore, we show simulation results of utilizing SCoT for feature extraction in a BCI application. These results indicate that CSPVARICA and correct regularization can significantly improve MI classification. While SCoT was mainly designed for application in BCIs, it contains useful tools for other areas of neuroscience. SCoT is a software package that (1) brings combined source decomposition and connectivtiy estimation to the open Python platform, and (2) offers tools for single-trial connectivity estimation. The source code is released under the MIT license and is available online at github.com/SCoT-dev/SCoT. PMID:24653694

Molecular cloning and functional expression of a stress-induced multifunctional O-methyltransferase with pinosylvin methyltransferase activity from Scots pine (Pinus sylvestris L.).

PubMed

Chiron, H; Drouet, A; Claudot, A C; Eckerskorn, C; Trost, M; Heller, W; Ernst, D; Sandermann, H

2000-12-01

Formation of pinosylvin (PS) and pinosylvin 3-O-monomethyl ether (PSM), as well as the activities of stilbene synthase (STS) and S-adenosyl-1-methionine (SAM):pinosylvin O-methyltransferase (PMT), were induced strongly in needles of Scots pine seedlings upon ozone treatment, as well as in cell suspension cultures of Scots pine upon fungal elicitation. A SAM-dependent PMT protein was purified and partially characterised. A cDNA encoding PMT was isolated from an ozone-induced Scots pine cDNA library. Southern blot analysis of the genomic DNA suggested the presence of a gene family. The deduced protein sequence showed the typical highly conserved regions of O-methyltransferases (OMTs), and average identities of 20-56% to known OMTs. PMT expressed in Escherichia coli corresponded to that of purified PMT (40 kDa) from pine cell cultures. The recombinant enzyme catalysed the methylation of PS, caffeic acid, caffeoyl-CoA and quercetin. Several other substances, such as astringenin, resveratrol, 5-OH-ferulic acid, catechol and luteolin, were also methylated. Recombinant PMT thus had a relatively broad substrate specificity. Treatment of 7-year old Scots pine trees with ozone markedly increased the PMT mRNA level. Our results show that PMT represents a new SAM-dependent OMT for the methylation of stress-induced pinosylvin in Scots pine needles.

[Post-logging organic matter recovery in forest ecosystems of eastern Baikal region].

PubMed

Vedrova, E F; Mukhortova, L V; Ivanov, V V; Krivobokov, L V; Boloneva, M V

2010-01-01

The dynamics of organic matter accumulated in the soil and main vegetation elements was analyzed for post-logging forest ecosystem succession series in eastern Baikal region. The phytomass was found to allocate up 63 and 50% of carbon in undisturbed Scots pine and fir stands, respectively. The post-logging phytomass contribution to the total carbon pool appeared to decrease down to 16% in Scots pine and 6% in fir stands. In Scots pine stands, carbon storage was determined to account for almost 70% of the initial carbon 60 years after logging. In 50- to 55-year-old fir stands, carbon recovered its initial pool only by 10%. Soil carbon recorded in recently logged Scots pine and fir sites appeared to be 5 and 16 times that accumulated in the phytomass, respectively. The ratio between phytomass carbon and soil organic matter recovered back to the prelogging level in Scots pine stands by the age of 50-60 years. While phytomass carbon also increased in fir stand of the same age, it did not reach the level of the control stand.

Developmental Changes in Scots Pine Transcriptome during Heartwood Formation1[OPEN

PubMed Central

Paasela, Tanja; Harju, Anni; Paulin, Lars; Auvinen, Petri; Kärkkäinen, Katri

2016-01-01

Scots pine (Pinus sylvestris L.) wood is desired in woodworking industries due to its favorable timber characteristics and natural durability that is contributed by heartwood extractives. It has been discussed whether the Scots pine heartwood extractives (mainly stilbenes and resin acids) are synthesized in the cells of the transition zone between sapwood and heartwood, or if they are transported from the sapwood. Timing of heartwood formation during the yearly cycle has also not been unambiguously defined. We measured steady-state mRNA levels in Scots pine transition zone and sapwood using RNA sequencing. Year-round expression profiles of selected transcripts were further investigated by quantitative RT-PCR. Differentially accumulating transcripts suggest that, of the Scots pine heartwood extractives, stilbenes are synthesized in situ in the transition zone and gain their carbon-skeletons from Suc and triglycerides. Resin acids, on the other hand, are synthesized early in the spring mainly in the sapwood, meaning that they must be transported to the heartwood transition zone. Heartwood formation is marked by programmed cell death that occurs during the summer months in the transition zone. PMID:27600814

The effects of multiple metal contamination on ectomycorrhizal Scots pine (Pinus sylvestris) seedlings.

PubMed

Hartley, J; Cairney, J W; Freestone, P; Woods, C; Meharg, A A

1999-09-01

Experiments were conducted to investigate the effects of single and multiple metal contamination (Cd, Pb, Zn, Sb, Cu) on Scots pine seedlings colonised by ectomycorrhizal (ECM) fungi from natural soil inoculum. Seedlings were grown in either contaminated field soil from the site of a chemical accident, soils amended with five metals contaminating the site, or in soil from an uncontaminated control site. Although contaminated and metal-amended soil significantly inhibited root and shoot growth of the Scots pine seedlings, total root tip density was not affected. Of the five metals tested in amended soils, Cd was the most toxic to ECM Scots pine. Field-contaminated soil had a toxic effect on ECM fungi associated with Scots pine seedlings and caused shifts in ECM species composition on ECM seedlings. When compared to soils amended with only one metal, soils amended with a combination of all five metals tested had lower relative toxicity and less accumulation of Pb, Zn and Sb into seedlings. This would indicate that the toxicity of multiple metal contamination cannot be predicted from the individual toxicity of the metals investigated.

Insight into pattern of codon biasness and nucleotide base usage in serotonin receptor gene family from different mammalian species.

PubMed

Dass, J Febin Prabhu; Sudandiradoss, C

2012-07-15

5-HT (5-Hydroxy-tryptamine) or serotonin receptors are found both in central and peripheral nervous system as well as in non-neuronal tissues. In the animal and human nervous system, serotonin produces various functional effects through a variety of membrane bound receptors. In this study, we focus on 5-HT receptor family from different mammals and examined the factors that account for codon and nucleotide usage variation. A total of 110 homologous coding sequences from 11 different mammalian species were analyzed using relative synonymous codon usage (RSCU), correspondence analysis (COA) and hierarchical cluster analysis together with nucleotide base usage frequency of chemically similar amino acid codons. The mean effective number of codon (ENc) value of 37.06 for 5-HT(6) shows very high codon bias within the family and may be due to high selective translational efficiency. The COA and Spearman's rank correlation reveals that the nucleotide compositional mutation bias as the major factors influencing the codon usage in serotonin receptor genes. The hierarchical cluster analysis suggests that gene function is another dominant factor that affects the codon usage bias, while species is a minor factor. Nucleotide base usage was reported using Goldman, Engelman, Stietz (GES) scale reveals the presence of high uracil (>45%) content at functionally important hydrophobic regions. Our in silico approach will certainly help for further investigations on critical inference on evolution, structure, function and gene expression aspects of 5-HT receptors family which are potential antipsychotic drug targets. Copyright © 2012 Elsevier B.V. All rights reserved.

Through Trial and Error: Learning and Adaptation in the English Tactical System from Bannockburn to Poitiers

DTIC Science & Technology

2014-06-13

continue to conduct irregular warfare through raids and ambushes. As the English vanguard approached, leery of attack from Scots in the wooded area... Scots , did not enter the wood line of New Park either. Robert the Bruce had successfully avoided battle with the English and repelled their...pulling back, to force the Scots further into the field so they could not retreat easily into the wood line, but his idea was challenged by a

Freedom’s Gate: The Southern Insurgency in the American Revolution, 1770-1781

DTIC Science & Technology

2014-01-01

of Loyalist militia units raised in the South. 3 Excepting Ferguson, a Scot serving in the British army, all of them were Americans. His ultimatum to...believed the backcountry of the Carolinas occupied by "a Sett of the most lowest vile crew breathing- Scot -Irish Presbyterians from the North of Ireland...informally in houses, hams, and often out in open. After 1666, the British army, given lists of Scots not attending Anglican services, literally began

Radiation exposure in the remote period after the Chernobyl accident caused oxidative stress and genetic effects in Scots pine populations

NASA Astrophysics Data System (ADS)

Volkova, Polina Yu.; Geras'Kin, Stanislav A.; Kazakova, Elizaveta A.

2017-02-01

Even 30 years after the Chernobyl accident, biological effects of irradiation are observed in the chronically exposed Scots pine populations. Chronic radiation exposure at dose rates above 50 mGy•yr-1 caused oxidative stress and led to the increase of antioxidants concentrations in these populations. Genetic variability was examined for 6 enzymes and 14 enzymatic loci of 6 Scots pine populations. Dose rates over 10 mGy•yr-1 caused the increased frequency of mutations and changes in genetic structure of Scots pine populations. However, the same dose rates had no effect on enzymatic activities. The results indicate that even relatively low dose rates of radiation can be considered as an ecological factor which should be taken into account for ecological management and radiation protection of biota species.

Development concepts of a Smart Cyber Operating Theater (SCOT) using ORiN technology.

PubMed

Okamoto, Jun; Masamune, Ken; Iseki, Hiroshi; Muragaki, Yoshihiro

2018-02-23

Currently, networking has not progressed in the treatment room. Almost every medical device in the treatment room operates as a stand-alone device. In this project, we aim to develop a networked operating room called "Smart Cyber Operating Theater (SCOT)". Medical devices are connected using Open Resource interface for the Network (ORiN) technology. In this paper, we describe the concept of the SCOT project. SCOT is integrated using the communication interface ORiN, which was originally developed for industry. One feature of ORiN is that the system can be constructed flexibly. ORiN creates abstracts of the same type of devices and increases the robustness of the system for device exchange. By using ORiN technology, we are developing new applications, such as decision-making navigation or a precision guided treatment system.

The proto-oncogene KRAS and BRAF profiles and some clinical characteristics in colorectal cancer in the Turkish population.

PubMed

Ozen, Filiz; Ozdemir, Semra; Zemheri, Ebru; Hacimuto, Gizem; Silan, Fatma; Ozdemir, Ozturk

2013-02-01

The aim of the current study was to investigate the prevalence and predictive significance of the KRAS and BRAF mutations in Turkish patients with colorectal cancer (CRC). Totally, 53 fresh tumoral tissue specimens were investigated in patients with CRC. All specimens were obtained during routine surgery of patients who were histopathologically diagnosed and genotyped for common KRAS and BRAF point mutations. After DNA extraction, the target mutations were analyzed using the AutoGenomics INFINITI(®) assay, and some samples were confirmed by quantitative real-time polymerase chain reaction fluorescence melting curve analyses. KRAS mutations were found in 26 (49.05%) CRC samples. Twenty-seven samples (50.95%) had wild-type profiles for KRAS codon 12, 13, and 61 in the current cohort. In 17 (65.38%) samples, codon 12; in 7 (26.93%) samples, codon 13; and in 2 (7.69%) samples, codon 61 were found to be mutated, particularly in grade 2 of tumoral tissues. No point mutation was detected in BRAF codon Val600Glu for the studied CRC patients. Our study, based on a representative collection of human CRC tumors, indicates that KRAS gene mutations were detected in 49.05% of the samples, and the most frequent mutation was in the G12D codon. Results also showed that codons 12 and 13 of KRAS are relatively frequently without BRAF mutation in a CRC cohort from the Turkish population.

Impregnation of Scots pine and beech with tannin solutions: effect of viscosity and wood anatomy in wood infiltration.

PubMed

Tondi, G; Thevenon, M F; Mies, B; Standfest, G; Petutschnigg, A; Wieland, S

The impregnation process of Scots pine and beech samples with tannin solutions was investigated. The two materials involved in the process (impregnation solution and wood samples) are studied in depth. Viscosity of mimosa tannin solutions and the anatomical aspect of beech and Scots pine were analysed and correlated. The viscosity of tannin solutions presents a non-newtonian behaviour when its pH level increases, and in the case of addition of hexamine as a hardener, the crosslinking of the flavonoids turns out to be of great importance. During the impregnation of Scots pine ( Pinus sylvestris L.) and beech ( Fagus sylvatica L.), the liquid and solid uptakes were monitored while taking into consideration the different conditions of the impregnation process. This method allowed to identify the best conditions needed in order to get a successful preservative uptake for each wooden substrate. The penetration mechanism within the wood of both species was revealed with the aid of a microscopic analysis. Scots pine is impregnated through the tracheids in the longitudinal direction and through parenchyma rays in the radial direction, whereas in beech, the penetration occurs almost completely through longitudinal vessels.

Developmental Changes in Scots Pine Transcriptome during Heartwood Formation.

PubMed

Lim, Kean-Jin; Paasela, Tanja; Harju, Anni; Venäläinen, Martti; Paulin, Lars; Auvinen, Petri; Kärkkäinen, Katri; Teeri, Teemu H

2016-11-01

Scots pine (Pinus sylvestris L.) wood is desired in woodworking industries due to its favorable timber characteristics and natural durability that is contributed by heartwood extractives. It has been discussed whether the Scots pine heartwood extractives (mainly stilbenes and resin acids) are synthesized in the cells of the transition zone between sapwood and heartwood, or if they are transported from the sapwood. Timing of heartwood formation during the yearly cycle has also not been unambiguously defined. We measured steady-state mRNA levels in Scots pine transition zone and sapwood using RNA sequencing. Year-round expression profiles of selected transcripts were further investigated by quantitative RT-PCR. Differentially accumulating transcripts suggest that, of the Scots pine heartwood extractives, stilbenes are synthesized in situ in the transition zone and gain their carbon-skeletons from Suc and triglycerides. Resin acids, on the other hand, are synthesized early in the spring mainly in the sapwood, meaning that they must be transported to the heartwood transition zone. Heartwood formation is marked by programmed cell death that occurs during the summer months in the transition zone. © 2016 American Society of Plant Biologists. All Rights Reserved.

High-accuracy Aspheric X-ray Mirror Metrology Using Software Configurable Optical Test System/deflectometry

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

Huang, Run; Su, Peng; Burge, James H.

The Software Configurable Optical Test System (SCOTS) uses deflectometry to measure surface slopes of general optical shapes without the need for additional null optics. Careful alignment of test geometry and calibration of inherent system error improve the accuracy of SCOTS to a level where it competes with interferometry. We report a SCOTS surface measurement of an off-axis superpolished elliptical x-ray mirror that achieves <1 nm<1 nm root-mean-square accuracy for the surface measurement with low-order term included.

Air Base Attacks and Defensive Counters: Historical Lessons and Future Challenges

DTIC Science & Technology

2015-01-01

I. Reid, Murray Scot Tanner, and Barry Wilson, A Question of Balance: Political Context and Military Aspects of the China–Taiwan Dispute, Santa...this case, Ramstein AB, Germany) placed fighter shelters on loops in woods . Although the shelters are clearly visible from directly overhead in...this image, placing them in woods would have made target acquisition and attack more difficult for Warsaw Pact fighters. That is because, in the 1970s

Innovative approach for transcriptomic analysis of obligate intracellular pathogen: selective capture of transcribed sequences of Ehrlichia ruminantium

PubMed Central

2009-01-01

Background Whole genome transcriptomic analysis is a powerful approach to elucidate the molecular mechanisms controlling the pathogenesis of obligate intracellular bacteria. However, the major hurdle resides in the low quantity of prokaryotic mRNAs extracted from host cells. Our model Ehrlichia ruminantium (ER), the causative agent of heartwater, is transmitted by tick Amblyomma variegatum. This bacterium affects wild and domestic ruminants and is present in Sub-Saharan Africa and the Caribbean islands. Because of its strictly intracellular location, which constitutes a limitation for its extensive study, the molecular mechanisms involved in its pathogenicity are still poorly understood. Results We successfully adapted the SCOTS method (Selective Capture of Transcribed Sequences) on the model Rickettsiales ER to capture mRNAs. Southern Blots and RT-PCR revealed an enrichment of ER's cDNAs and a diminution of ribosomal contaminants after three rounds of capture. qRT-PCR and whole-genome ER microarrays hybridizations demonstrated that SCOTS method introduced only a limited bias on gene expression. Indeed, we confirmed the differential gene expression between poorly and highly expressed genes before and after SCOTS captures. The comparative gene expression obtained from ER microarrays data, on samples before and after SCOTS at 96 hpi was significantly correlated (R2 = 0.7). Moreover, SCOTS method is crucial for microarrays analysis of ER, especially for early time points post-infection. There was low detection of transcripts for untreated samples whereas 24% and 70.7% were revealed for SCOTS samples at 24 and 96 hpi respectively. Conclusions We conclude that this SCOTS method has a key importance for the transcriptomic analysis of ER and can be potentially used for other Rickettsiales. This study constitutes the first step for further gene expression analyses that will lead to a better understanding of both ER pathogenicity and the adaptation of obligate intracellular bacteria to their environment. PMID:20034374

Rous Sarcoma Virus RNA Stability Element Inhibits Deadenylation of mRNAs with Long 3′UTRs

PubMed Central

Balagopal, Vidya; Beemon, Karen L.

2017-01-01

All retroviruses use their full-length primary transcript as the major mRNA for Group-specific antigen (Gag) capsid proteins. This results in a long 3′ untranslated region (UTR) downstream of the termination codon. In the case of Rous sarcoma virus (RSV), there is a 7 kb 3′UTR downstream of the gag terminator, containing the pol, env, and src genes. mRNAs containing long 3′UTRs, like those with premature termination codons, are frequently recognized by the cellular nonsense-mediated mRNA decay (NMD) machinery and targeted for degradation. To prevent this, RSV has evolved an RNA stability element (RSE) in the RNA immediately downstream of the gag termination codon. This 400-nt RNA sequence stabilizes premature termination codons (PTCs) in gag. It also stabilizes globin mRNAs with long 3′UTRs, when placed downstream of the termination codon. It is not clear how the RSE stabilizes the mRNA and prevents decay. We show here that the presence of RSE inhibits deadenylation severely. In addition, the RSE also impairs decapping (DCP2) and 5′-3′ exonucleolytic (XRN1) function in knockdown experiments in human cells. PMID:28763028

Stem Cell Ophthalmology Treatment Study (SCOTS) for retinal and optic nerve diseases: a case report of improvement in relapsing auto-immune optic neuropathy.

PubMed

Weiss, Jeffrey N; Levy, Steven; Benes, Susan C

2015-09-01

We present the results from a patient with relapsing optic neuropathy treated within the Stem Cell Ophthalmology Treatment Study (SCOTS). SCOTS is an Institutional Review Board approved clinical trial and has become the largest ophthalmology stem cell study registered at the National Institutes of Health to date (www.clinicaltrials.gov Identifier NCT 01920867). SCOTS utilizes autologous bone marrow-derived stem cells (BMSCs) for treatment of retinal and optic nerve diseases. Pre-treatment and post-treatment comprehensive eye exams of a 54 year old female patient were performed both at the Florida Study Center, USA and at The Eye Center of Columbus, USA. As a consequence of a relapsing optic neuritis, the patient's previously normal visual acuity decreased to between 20/350 and 20/400 in the right eye and to 20/70 in the left eye. Significant visual field loss developed bilaterally. The patient underwent a right eye vitrectomy with injection of BMSCs into the optic nerve of the right eyeand retrobulbar, subtenon and intravitreal injection of BMSCs in the left eye. At 15 months after SCOTS treatment, the patient's visual acuity had improved to 20/150 in the right eye and 20/20 in the left eye. Bilateral visual fields improved markedly. Both macular thickness and fast retinal nerve fiber layer thickness were maximally improved at 3 and 6 months after SCOTS treatment. The patient also reduced her mycophenylate dose from 1,500 mg per day to 500 mg per day and required no steroid pulse therapy during the 15-month follow up.

Effects of tRNA modification on translational accuracy depend on intrinsic codon-anticodon strength.

PubMed

Manickam, Nandini; Joshi, Kartikeya; Bhatt, Monika J; Farabaugh, Philip J

2016-02-29

Cellular health and growth requires protein synthesis to be both efficient to ensure sufficient production, and accurate to avoid producing defective or unstable proteins. The background of misreading error frequency by individual tRNAs is as low as 2 × 10(-6) per codon but is codon-specific with some error frequencies above 10(-3) per codon. Here we test the effect on error frequency of blocking post-transcriptional modifications of the anticodon loops of four tRNAs in Escherichia coli. We find two types of responses to removing modification. Blocking modification of tRNA(UUC)(Glu) and tRNA(QUC)(Asp) increases errors, suggesting that the modifications act at least in part to maintain accuracy. Blocking even identical modifications of tRNA(UUU)(Lys) and tRNA(QUA)(Tyr) has the opposite effect of decreasing errors. One explanation could be that the modifications play opposite roles in modulating misreading by the two classes of tRNAs. Given available evidence that modifications help preorder the anticodon to allow it to recognize the codons, however, the simpler explanation is that unmodified 'weak' tRNAs decode too inefficiently to compete against cognate tRNAs that normally decode target codons, which would reduce the frequency of misreading. © The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.

Value of Information and Information Services

DOT National Transportation Integrated Search

1975-10-01

The report describes the salient features of the SCOT (Simulation of Corridor Traffic) model and a successful calibration and validation. SCOT is a computer model that may be applied to an urban traffic corridor and will simulate vehicular traffic on...

B cell Variable genes have evolved their codon usage to focus the targeted patterns of somatic mutation on the complementarity determining regions

PubMed Central

Saini, Jasmine; Hershberg, Uri

2015-01-01

The exceptional ability of B cells to diversify through somatic mutation and improve affinity of the repertoire towards the antigens is the cornerstone of adaptive immunity. Somatic mutation is not evenly distributed and exhibits certain micro-sequence specificities. We show here that the combination of somatic mutation targeting and the codon usage in human B cell receptor (BCR) Variable (V) genes create expected patterns of mutation and post mutation changes that are focused on their complementarity determining regions (CDR). T cell V genes are also skewed in targeting mutations but to a lesser extent and are lacking the codon usage bias observed in BCRs. This suggests that the observed skew in T cell receptors is due to their amino acid usage, which is similar to that of BCRs. The mutation targeting and the codon bias allow B cell CDRs to diversify by specifically accumulating nonconservative changes. We counted the distribution of mutations to CDR in 4 different human datasets. In all four cases we found that the number of actual mutations in the CDR correlated significantly with the V gene mutation biases to the CDR predicted by our models. Finally, it appears that the mutation bias in V genes indeed relates to their long-term survival in actual human repertoires. We observed that resting repertoires of B cells overexpressed V genes that were especially biased towards focused mutation and change in the CDR. This bias in V gene usage was somewhat relaxed at the height of the immune response to a vaccine, presumably because of the need for a wider diversity in a primary response. However, older patients did not retain this flexibility and were biased towards using only highly skewed V genes at all stages of their response. PMID:25660968

B cell variable genes have evolved their codon usage to focus the targeted patterns of somatic mutation on the complementarity determining regions.

PubMed

Saini, Jasmine; Hershberg, Uri

2015-05-01

The exceptional ability of B cells to diversify through somatic mutation and improve affinity of the repertoire toward the antigens is the cornerstone of adaptive immunity. Somatic mutation is not evenly distributed and exhibits certain micro-sequence specificities. We show here that the combination of somatic mutation targeting and the codon usage in human B cell receptor (BCR) Variable (V) genes create expected patterns of mutation and post mutation changes that are focused on their complementarity determining regions (CDR). T cell V genes are also skewed in targeting mutations but to a lesser extent and are lacking the codon usage bias observed in BCRs. This suggests that the observed skew in T cell receptors is due to their amino acid usage, which is similar to that of BCRs. The mutation targeting and the codon bias allow B cell CDRs to diversify by specifically accumulating nonconservative changes. We counted the distribution of mutations to CDR in 4 different human datasets. In all four cases we found that the number of actual mutations in the CDR correlated significantly with the V gene mutation biases to the CDR predicted by our models. Finally, it appears that the mutation bias in V genes indeed relates to their long-term survival in actual human repertoires. We observed that resting repertoires of B cells overexpressed V genes that were especially biased toward focused mutation and change in the CDR. This bias in V gene usage was somewhat relaxed at the height of the immune response to a vaccine, presumably because of the need for a wider diversity in a primary response. However, older patients did not retain this flexibility and were biased toward using only highly skewed V genes at all stages of their response. Copyright © 2015 Elsevier Ltd. All rights reserved.

Genetics Home Reference: succinyl-CoA:3-ketoacid CoA transferase deficiency

MedlinePlus

... more common in particular ethnic groups? Genetic Changes Mutations in the OXCT1 gene cause SCOT deficiency. The ... as during illness or when exercising. OXCT1 gene mutations result in the production of a SCOT enzyme ...

Growth, aboveground biomass, and nutrient concentration of young Scots pine and lodgepole pine in oil shale post-mining landscapes in Estonia.

PubMed

Kuznetsova, Tatjana; Tilk, Mari; Pärn, Henn; Lukjanova, Aljona; Mandre, Malle

2011-12-01

The investigation was carried out in 8-year-old Scots pine (Pinus sylvestris L.) and lodgepole pine (Pinus contorta var. latifolia Engelm.) plantations on post-mining area, Northeast Estonia. The aim of the study was to assess the suitability of lodgepole pine for restoration of degraded lands by comparing the growth, biomass, and nutrient concentration of studied species. The height growth of trees was greater in the Scots pine stand, but the tree aboveground biomass was slightly larger in the lodgepole pine stand. The aboveground biomass allocation to the compartments did not differ significantly between species. The vertical distribution of compartments showed that 43.2% of the Scots pine needles were located in the middle layer of the crown, while 58.5% of the lodgepole pine needles were in the lowest layer of the crown. The largest share of the shoots and stem of both species was allocated to the lowest layer of the crown. For both species, the highest NPK concentrations were found in the needles and the lowest in the stems. On the basis of the present study results, it can be concluded that the early growth of Scots pine and lodgepole pine on oil shale post-mining landscapes is similar.

BATTLE (Biomarker-based Approach of Targeted Therapy for Lung Cancer Elimination)

DTIC Science & Technology

2012-04-01

Wistuba,Ignacio Ivan Woods ,Denise M Wu,Huakang Xue,Yuwen Yang,Bijun Yin,Ming Zhang,Guangcheng Zhang,Li Army...Oncol. 2009; 27: 271–8. 127 Ishii Y, Rex M, Scotting PJ et al. Region-specific expression of chicken Sox2 in the developing gut and lung epithelium... Wood ER, Shewchuk L, Hassel A, et al. Discovery of an in- hibitor of insulin-like growth factor 1 receptor activation: implications for cellular potency

A Functional and Structural Mongolian Scots Pine (Pinus sylvestris var. mongolica) Model Integrating Architecture, Biomass and Effects of Precipitation

PubMed Central

Wang, Feng; Letort, Véronique; Lu, Qi; Bai, Xuefeng; Guo, Yan; de Reffye, Philippe; Li, Baoguo

2012-01-01

Mongolian Scots pine (Pinus sylvestris var. mongolica) is one of the principal tree species in the network of Three-North Shelterbelt for windbreak and sand stabilisation in China. The functions of shelterbelts are highly correlated with the architecture and eco-physiological processes of individual tree. Thus, model-assisted analysis of canopy architecture and function dynamic in Mongolian Scots pine is of value for better understanding its role and behaviour within shelterbelt ecosystems in these arid and semiarid regions. We present here a single-tree functional and structural model, derived from the GreenLab model, which is adapted for young Mongolian Scots pines by incorporation of plant biomass production, allocation, allometric rules and soil water dynamics. The model is calibrated and validated based on experimental measurements taken on Mongolian Scots pines in 2007 and 2006 under local meteorological conditions. Measurements include plant biomass, topology and geometry, as well as soil attributes and standard meteorological data. After calibration, the model allows reconstruction of three-dimensional (3D) canopy architecture and biomass dynamics for trees from one- to six-year-old at the same site using meteorological data for the six years from 2001 to 2006. Sensitivity analysis indicates that rainfall variation has more influence on biomass increment than on architecture, and the internode and needle compartments and the aboveground biomass respond linearly to increases in precipitation. Sensitivity analysis also shows that the balance between internode and needle growth varies only slightly within the range of precipitations considered here. The model is expected to be used to investigate the growth of Mongolian Scots pines in other regions with different soils and climates. PMID:22927982

Impact of Peripheral Ketolytic Deficiency on Hepatic Ketogenesis and Gluconeogenesis during the Transition to Birth*

PubMed Central

Cotter, David G.; Ercal, Baris; d'Avignon, D. André; Dietzen, Dennis J.; Crawford, Peter A.

2013-01-01

Preservation of bioenergetic homeostasis during the transition from the carbohydrate-laden fetal diet to the high fat, low carbohydrate neonatal diet requires inductions of hepatic fatty acid oxidation, gluconeogenesis, and ketogenesis. Mice with loss-of-function mutation in the extrahepatic mitochondrial enzyme CoA transferase (succinyl-CoA:3-oxoacid CoA transferase, SCOT, encoded by nuclear Oxct1) cannot terminally oxidize ketone bodies and develop lethal hyperketonemic hypoglycemia within 48 h of birth. Here we use this model to demonstrate that loss of ketone body oxidation, an exclusively extrahepatic process, disrupts hepatic intermediary metabolic homeostasis after high fat mother's milk is ingested. Livers of SCOT-knock-out (SCOT-KO) neonates induce the expression of the genes encoding peroxisome proliferator-activated receptor γ co-activator-1a (PGC-1α), phosphoenolpyruvate carboxykinase (PEPCK), pyruvate carboxylase, and glucose-6-phosphatase, and the neonate's pools of gluconeogenic alanine and lactate are each diminished by 50%. NMR-based quantitative fate mapping of 13C-labeled substrates revealed that livers of SCOT-KO newborn mice synthesize glucose from exogenously administered pyruvate. However, the contribution of exogenous pyruvate to the tricarboxylic acid cycle as acetyl-CoA is increased in SCOT-KO livers and is associated with diminished terminal oxidation of fatty acids. After mother's milk provokes hyperketonemia, livers of SCOT-KO mice diminish de novo hepatic β-hydroxybutyrate synthesis by 90%. Disruption of β-hydroxybutyrate production increases hepatic NAD+/NADH ratios 3-fold, oxidizing redox potential in liver but not skeletal muscle. Together, these results indicate that peripheral ketone body oxidation prevents hypoglycemia and supports hepatic metabolic homeostasis, which is critical for the maintenance of glycemia during the adaptation to birth. PMID:23689508

Imaging spectroscopy in soil-water based site suitability assessment for artificial regeneration to Scots pine

NASA Astrophysics Data System (ADS)

Middleton, Maarit; Närhi, Paavo; Sutinen, Raimo

In a humid northern boreal climate, the success rate of artificial regeneration to Scots pine ( Pinus sylvestris L.) can be improved by including a soil water content (SWC) based assessment of site suitability in the reforestation planning process. This paper introduces an application of airborne visible-near-infrared imaging spectroscopic data to identify suitable subregions of forest compartments for the low SWC-tolerant Scots pine. The spatial patterns of understorey plant species communities, recorded by the AISA (Airborne Imaging Spectrometer for Applications) sensor, were demonstrated to be dependant on the underlying SWC. According to the nonmetric multidimensional scaling and correlation results twelve understorey species were found to be most abundant on sites with high soil SWCs. The abundance of bare soil, rocks and abundance of more than ten species indicated low soil SWCs. The spatial patterns of understorey are attributed to time-stability of the underlying SWC patterns. A supervised artificial neural network (radial basis functional link network, probabilistic neural network) approach was taken to classify AISA imaging spectrometer data with dielectric (as a measure volumetric SWC) ground referencing into regimes suitable and unsuitable for Scots pine. The accuracy assessment with receiver operating characteristics curves demonstrated a maximum of 74.1% area under the curve values which indicated moderate success of the NN modelling. The results signified the importance of the training set's quality, adequate quantity (>2.43 points/ha) and NN algorithm selection over the NN algorithm training parameter optimization to perfection. This methodology for the analysis of site suitability of Scots pine can be recommended, especially when artificial regeneration of former mixed wood Norway spruce ( Picea abies L. Karst) - downy birch ( Betula pubenscens Ehrh.) stands is being considered, so that artificially regenerated areas to Scots pine can be optimized for forestry purposes.

Genetic Diversity and Association of EST-SSR and SCoT Markers with Rust Traits in Orchardgrass (Dactylis glomerata L.).

PubMed

Yan, Haidong; Zhang, Yu; Zeng, Bing; Yin, Guohua; Zhang, Xinquan; Ji, Yang; Huang, Linkai; Jiang, Xiaomei; Liu, Xinchun; Peng, Yan; Ma, Xiao; Yan, Yanhong

2016-01-08

Orchardgrass (Dactylis glomerata L.), is a well-known perennial forage species; however, rust diseases have caused a noticeable reduction in the quality and production of orchardgrass. In this study, genetic diversity was assessed and the marker-trait associations for rust were examined using 18 EST-SSR and 21 SCoT markers in 75 orchardgrass accessions. A high level of genetic diversity was detected in orchardgrass with an average genetic diversity index of 0.369. For the EST-SSR and SCoT markers, 164 and 289 total bands were obtained, of which 148 (90.24%) and 272 (94.12%) were polymorphic, respectively. Results from an AMOVA analysis showed that more genetic variance existed within populations (87.57%) than among populations (12.43%). Using a parameter marker index, the efficiencies of the EST-SSR and SCoT markers were compared to show that SCoTs have higher marker efficiency (8.07) than EST-SSRs (4.82). The results of a UPGMA cluster analysis and a STRUCTURE analysis were both correlated with the geographic distribution of the orchardgrass accessions. Linkage disequilibrium analysis revealed an average r² of 0.1627 across all band pairs, indicating a high extent of linkage disequilibrium in the material. An association analysis between the rust trait and 410 bands from the EST-SSR and SCoT markers using TASSEL software revealed 20 band panels were associated with the rust trait in both 2011 and 2012. The 20 bands obtained from association analysis could be used in breeding programs for lineage selection to prevent great losses of orchardgrass caused by rust, and provide valuable information for further association mapping using this collection of orchardgrass.

Successful adaptation to ketosis by mice with tissue-specific deficiency of ketone body oxidation

PubMed Central

Cotter, David G.; Schugar, Rebecca C.; Wentz, Anna E.; André d'Avignon, D.

2013-01-01

During states of low carbohydrate intake, mammalian ketone body metabolism transfers energy substrates originally derived from fatty acyl chains within the liver to extrahepatic organs. We previously demonstrated that the mitochondrial enzyme coenzyme A (CoA) transferase [succinyl-CoA:3-oxoacid CoA transferase (SCOT), encoded by nuclear Oxct1] is required for oxidation of ketone bodies and that germline SCOT-knockout (KO) mice die within 48 h of birth because of hyperketonemic hypoglycemia. Here, we use novel transgenic and tissue-specific SCOT-KO mice to demonstrate that ketone bodies do not serve an obligate energetic role within highly ketolytic tissues during the ketogenic neonatal period or during starvation in the adult. Although transgene-mediated restoration of myocardial CoA transferase in germline SCOT-KO mice is insufficient to prevent lethal hyperketonemic hypoglycemia in the neonatal period, mice lacking CoA transferase selectively within neurons, cardiomyocytes, or skeletal myocytes are all viable as neonates. Like germline SCOT-KO neonatal mice, neonatal mice with neuronal CoA transferase deficiency exhibit increased cerebral glycolysis and glucose oxidation, and, while these neonatal mice exhibit modest hyperketonemia, they do not develop hypoglycemia. As adults, tissue-specific SCOT-KO mice tolerate starvation, exhibiting only modestly increased hyperketonemia. Finally, metabolic analysis of adult germline Oxct1+/− mice demonstrates that global diminution of ketone body oxidation yields hyperketonemia, but hypoglycemia emerges only during a protracted state of low carbohydrate intake. Together, these data suggest that, at the tissue level, ketone bodies are not a required energy substrate in the newborn period or during starvation, but rather that integrated ketone body metabolism mediates adaptation to ketogenic nutrient states. PMID:23233542

Successful adaptation to ketosis by mice with tissue-specific deficiency of ketone body oxidation.

PubMed

Cotter, David G; Schugar, Rebecca C; Wentz, Anna E; d'Avignon, D André; Crawford, Peter A

2013-02-15

During states of low carbohydrate intake, mammalian ketone body metabolism transfers energy substrates originally derived from fatty acyl chains within the liver to extrahepatic organs. We previously demonstrated that the mitochondrial enzyme coenzyme A (CoA) transferase [succinyl-CoA:3-oxoacid CoA transferase (SCOT), encoded by nuclear Oxct1] is required for oxidation of ketone bodies and that germline SCOT-knockout (KO) mice die within 48 h of birth because of hyperketonemic hypoglycemia. Here, we use novel transgenic and tissue-specific SCOT-KO mice to demonstrate that ketone bodies do not serve an obligate energetic role within highly ketolytic tissues during the ketogenic neonatal period or during starvation in the adult. Although transgene-mediated restoration of myocardial CoA transferase in germline SCOT-KO mice is insufficient to prevent lethal hyperketonemic hypoglycemia in the neonatal period, mice lacking CoA transferase selectively within neurons, cardiomyocytes, or skeletal myocytes are all viable as neonates. Like germline SCOT-KO neonatal mice, neonatal mice with neuronal CoA transferase deficiency exhibit increased cerebral glycolysis and glucose oxidation, and, while these neonatal mice exhibit modest hyperketonemia, they do not develop hypoglycemia. As adults, tissue-specific SCOT-KO mice tolerate starvation, exhibiting only modestly increased hyperketonemia. Finally, metabolic analysis of adult germline Oxct1(+/-) mice demonstrates that global diminution of ketone body oxidation yields hyperketonemia, but hypoglycemia emerges only during a protracted state of low carbohydrate intake. Together, these data suggest that, at the tissue level, ketone bodies are not a required energy substrate in the newborn period or during starvation, but rather that integrated ketone body metabolism mediates adaptation to ketogenic nutrient states.

Fine root morphological adaptations in Scots pine, Norway spruce and silver birch along a latitudinal gradient in boreal forests.

PubMed

Ostonen, Ivika; Lõhmus, Krista; Helmisaari, Heljä-Sisko; Truu, Jaak; Meel, Signe

2007-11-01

Variability in short root morphology of the three main tree species of Europe's boreal forest (Norway spruce (Picea abies L. Karst.), Scots pine (Pinus sylvestris L.) and silver birch (Betula pendula Roth)) was investigated in four stands along a latitudinal gradient from northern Finland to southern Estonia. Silver birch and Scots pine were present in three stands and Norway spruce was present in all stands. For three fertile Norway spruce stands, fine root biomass and number of root tips per stand area or unit basal area were assessed from north to south. Principal component analysis indicated that short root morphology was significantly affected by tree species and site, which together explained 34.7% of the total variability. The range of variation in mean specific root area (SRA) was 51-74, 60-70 and 84-124 m(2) kg(-1) for Norway spruce, Scots pine and silver birch, respectively, and the corresponding ranges for specific root length were 37-47, 40-48 and 87-97 m g(-1). The range of variation in root tissue density of Norway spruce, Scots pine and silver birch was 113-182, 127-158 and 81-156 kg m(-3), respectively. Sensitivity of short root morphology to site conditions decreased in the order: Norway spruce > silver birch > Scots pine. Short root SRA increased with site fertility in all species. In Norway spruce, fine root biomass and number of root tips per m(2) decreased from north to south. The differences in morphological parameters among sites were significant but smaller than the site differences in fine root biomass and number of root tips.

A high-level prokaryotic expression system: synthesis of human interleukin 1 alpha and its receptor antagonist.

PubMed

Birikh, K R; Lebedenko, E N; Boni, I V; Berlin, Y A

1995-10-27

Synthetic intronless genes, coding for human interleukin 1 alpha (IL 1 alpha) and interleukin 1 receptor antagonist (IL1ra), have been expressed efficiently in a specially designed prokaryotic vector, pGMCE (a pGEM1 derivative), where the target gene forms the second part of a two-cistron system. The first part of the system is a translation enhancer-containing mini-cistron, whose termination codon overlaps the start codon of the target gene. In the case of the IL1 alpha gene, the high expression level is largely due to the direct efficient translation initiation at the second cistron, whereas with the IL1ra gene in the same system, the proximal translation initiation region (TIR) provides a high level of coupled expression of the target gene. Thus, pGMCE is a potentially versatile vector for direct prokaryotic expression.

Long-term exposure to enhanced UV-B radiation has no significant effects on growth or secondary compounds of outdoor-grown Scots pine and Norway spruce seedlings.

PubMed

Turtola, Satu; Sallas, Leena; Holopainen, Jarmo K; Julkunen-Tiitto, Riitta; Kainulainen, Pirjo

2006-11-01

The effects of long-term enhanced UV-B radiation on growth and secondary compounds of two conifer species were studied in an outdoor experiment. Scots pine (Pinus sylvestris) seedlings were exposed for two growing seasons and Norway spruce (Picea abies) seedlings for three growing seasons to supplemental UV-B radiation, corresponding to a 30% increase in ambient UV-B radiation. The experiment also included appropriate controls for ambient and increased UV-A radiation. Enhanced UV-B did not affect the growth of the conifer seedlings. In addition, neither the concentrations of terpenes and phenolics in the needles nor the concentrations of terpenes in the wood were affected. However, in the UV-A control treatment the concentrations of diterpenes in the wood of Scots pine decreased significantly compared to the ambient control. Apparently, a small increase in UV-B radiation has no significant effects on the secondary compounds and growth of Scots pine and Norway spruce seedlings.

Manufacture of Autologous CD34+ Selected Grafts in the NIAID-Sponsored HALT-MS and SCOT Multicenter Clinical Trials for Autoimmune Diseases.

PubMed

Keever-Taylor, Carolyn A; Heimfeld, Shelly; Steinmiller, Kaitlyn C; Nash, Richard A; Sullivan, Keith M; Czarniecki, Christine W; Granderson, Tomeka C; Goldstein, Julia S; Griffith, Linda M

2017-09-01

To ensure comparable grafts for autologous hematopoietic cell transplantation (HCT) in the National Institute of Allergy and Infectious Diseases-sponsored Investigational New Drug protocols for multiple sclerosis (HALT-MS) and systemic sclerosis (SCOT), a Drug Master File approach to control manufacture was implemented, including a common Master Production Batch Record and site-specific standard operating procedures with "Critical Elements." We assessed comparability of flow cytometry and controlled rate cryopreservation among sites and stability of cryopreserved grafts using hematopoietic progenitor cells (HPCs) from healthy donors. Hematopoietic Progenitor Cells, Apheresis-CD34+ Enriched, for Autologous Use (Auto-CD34 + HPC) graft specifications included ≥70% viable CD34 + cells before cryopreservation. For the 2 protocols, 110 apheresis collections were performed; 121 lots of Auto-CD34 + HPC were cryopreserved, and 107 of these (88.4%) met release criteria. Grafts were infused at a median of 25 days (range, 17 to 68) post-apheresis for HALT-MS (n = 24), and 25 days (range, 14 to 78) for SCOT (n = 33). Subjects received precryopreservation doses of a median 5.1 × 10 6 viable CD34 + cells/kg (range, 3.9 to 12.8)  for HALT-MS and 5.6 × 10 6 viable CD34 + cells/kg (range, 2.6 to 10.2) for SCOT. Recovery of granulocytes occurred at a median of 11 days (range, 9 to 15) post-HCT for HALT-MS and 10 days (range, 8 to 12) for SCOT, independent of CD34 + cell dose. Subjects received their last platelet transfusion at a median of 9 days (range, 6 to 16) for HALT-MS and 8 days (range, 6 to 23) for SCOT; higher CD34 + /kg doses were associated with faster platelet recovery. Stability testing of cryopreserved healthy donor CD34 + HPCs over 6 months of vapor phase liquid nitrogen storage demonstrated consistent 69% to 73% recovery of viable CD34 + cells. Manufacturing of Auto-CD34 + HPC for the HALT-MS and SCOT protocols was comparable across all sites and supportive for timely recovery of granulocytes and platelets. Published by Elsevier Inc.

A stochastic model of tree architecture and biomass partitioning: application to Mongolian Scots pines

PubMed Central

Wang, Feng; Kang, Mengzhen; Lu, Qi; Letort, Véronique; Han, Hui; Guo, Yan; de Reffye, Philippe; Li, Baoguo

2011-01-01

Background and Aims Mongolian Scots pine (Pinus sylvestris var. mongolica) is one of the principal species used for windbreak and sand stabilization in arid and semi-arid areas in northern China. A model-assisted analysis of its canopy architectural development and functions is valuable for better understanding its behaviour and roles in fragile ecosystems. However, due to the intrinsic complexity and variability of trees, the parametric identification of such models is currently a major obstacle to their evaluation and their validation with respect to real data. The aim of this paper was to present the mathematical framework of a stochastic functional–structural model (GL2) and its parameterization for Mongolian Scots pines, taking into account inter-plant variability in terms of topological development and biomass partitioning. Methods In GL2, plant organogenesis is determined by the realization of random variables representing the behaviour of axillary or apical buds. The associated probabilities are calibrated for Mongolian Scots pines using experimental data including means and variances of the numbers of organs per plant in each order-based class. The functional part of the model relies on the principles of source–sink regulation and is parameterized by direct observations of living trees and the inversion method using measured data for organ mass and dimensions. Key Results The final calibration accuracy satisfies both organogenetic and morphogenetic processes. Our hypothesis for the number of organs following a binomial distribution is found to be consistent with the real data. Based on the calibrated parameters, stochastic simulations of the growth of Mongolian Scots pines in plantations are generated by the Monte Carlo method, allowing analysis of the inter-individual variability of the number of organs and biomass partitioning. Three-dimensional (3D) architectures of young Mongolian Scots pines were simulated for 4-, 6- and 8-year-old trees. Conclusions This work provides a new method for characterizing tree structures and biomass allocation that can be used to build a 3D virtual Mongolian Scots pine forest. The work paves the way for bridging the gap between a single-plant model and a stand model. PMID:21062760

A stochastic model of tree architecture and biomass partitioning: application to Mongolian Scots pines.

PubMed

Wang, Feng; Kang, Mengzhen; Lu, Qi; Letort, Véronique; Han, Hui; Guo, Yan; de Reffye, Philippe; Li, Baoguo

2011-04-01

Mongolian Scots pine (Pinus sylvestris var. mongolica) is one of the principal species used for windbreak and sand stabilization in arid and semi-arid areas in northern China. A model-assisted analysis of its canopy architectural development and functions is valuable for better understanding its behaviour and roles in fragile ecosystems. However, due to the intrinsic complexity and variability of trees, the parametric identification of such models is currently a major obstacle to their evaluation and their validation with respect to real data. The aim of this paper was to present the mathematical framework of a stochastic functional-structural model (GL2) and its parameterization for Mongolian Scots pines, taking into account inter-plant variability in terms of topological development and biomass partitioning. In GL2, plant organogenesis is determined by the realization of random variables representing the behaviour of axillary or apical buds. The associated probabilities are calibrated for Mongolian Scots pines using experimental data including means and variances of the numbers of organs per plant in each order-based class. The functional part of the model relies on the principles of source-sink regulation and is parameterized by direct observations of living trees and the inversion method using measured data for organ mass and dimensions. The final calibration accuracy satisfies both organogenetic and morphogenetic processes. Our hypothesis for the number of organs following a binomial distribution is found to be consistent with the real data. Based on the calibrated parameters, stochastic simulations of the growth of Mongolian Scots pines in plantations are generated by the Monte Carlo method, allowing analysis of the inter-individual variability of the number of organs and biomass partitioning. Three-dimensional (3D) architectures of young Mongolian Scots pines were simulated for 4-, 6- and 8-year-old trees. This work provides a new method for characterizing tree structures and biomass allocation that can be used to build a 3D virtual Mongolian Scots pine forest. The work paves the way for bridging the gap between a single-plant model and a stand model.

Increasing the fidelity of noncanonical amino acid incorporation in cell-free protein synthesis.

PubMed

Gan, Qinglei; Fan, Chenguang

2017-11-01

Cell-free protein synthesis provides a robust platform for co-translational incorporation of noncanonical amino acid (ncAA) into proteins to facilitate biological studies and biotechnological applications. Recently, eliminating the activity of release factor 1 has been shown to increase ncAA incorporation in response to amber codons. However, this approach could promote mis-incorporation of canonical amino acids by near cognate suppression. We performed a facile protocol to remove near cognate tRNA isoacceptors of the amber codon from total tRNAs, and used the phosphoserine (Sep) incorporation system as validation. By manipulating codon usage of target genes and tRNA species introduced into the cell-free protein synthesis system, we increased the fidelity of Sep incorporation at a specific position. By removing three near cognate tRNA isoacceptors of the amber stop codon [tRNA Lys , tRNA Tyr , and tRNA Gln (CUG)] from the total tRNA, the near cognate suppression decreased by 5-fold without impairing normal protein synthesis in the cell-free protein synthesis system. Mass spectrometry analyses indicated that the fidelity of ncAA incorporation was improved. Removal of near cognate tRNA isoacceptors of the amber codon could increase ncAA incorporation fidelity towards the amber stop codon in release factor deficiency systems. We provide a general strategy to improve fidelity of ncAA incorporation towards stop, quadruplet and sense codons in cell-free protein synthesis systems. This article is part of a Special Issue entitled "Biochemistry of Synthetic Biology - Recent Developments" Guest Editor: Dr. Ilka Heinemann and Dr. Patrick O'Donoghue. Copyright © 2016 Elsevier B.V. All rights reserved.

Supply-Chain Optimization Template

NASA Technical Reports Server (NTRS)

Quiett, William F.; Sealing, Scott L.

2009-01-01

The Supply-Chain Optimization Template (SCOT) is an instructional guide for identifying, evaluating, and optimizing (including re-engineering) aerospace- oriented supply chains. The SCOT was derived from the Supply Chain Council s Supply-Chain Operations Reference (SCC SCOR) Model, which is more generic and more oriented toward achieving a competitive advantage in business.

Standard/Handbook for Multipactor Breakdown Prevention in Spacecraft Components

DTIC Science & Technology

2014-05-28

Axley, Transient-mode Multipactor Discharge, Phys. Plasmas, 2009, 16, n. 8, pp. 083502-1 – 083502-7. 11. D. Woode and J. Petit. ESTEC Working Paper 1556...Bearings Scot Lichty scot.r.lichty@lmco.com Lockheed Martin Sultan Ali Lilani sultan.lilani@integra- tech.com Integra - Tech Josh Lindley

CRISPR-STOP: gene silencing through base-editing-induced nonsense mutations.

PubMed

Kuscu, Cem; Parlak, Mahmut; Tufan, Turan; Yang, Jiekun; Szlachta, Karol; Wei, Xiaolong; Mammadov, Rashad; Adli, Mazhar

2017-07-01

CRISPR-Cas9-induced DNA damage may have deleterious effects at high-copy-number genomic regions. Here, we use CRISPR base editors to knock out genes by changing single nucleotides to create stop codons. We show that the CRISPR-STOP method is an efficient and less deleterious alternative to wild-type Cas9 for gene-knockout studies. Early stop codons can be introduced in ∼17,000 human genes. CRISPR-STOP-mediated targeted screening demonstrates comparable efficiency to WT Cas9, which indicates the suitability of our approach for genome-wide functional screenings.

Whole genome sequencing based characterization of extensively drug-resistant Mycobacterium tuberculosis isolates from Pakistan.

PubMed

Ali, Asho; Hasan, Zahra; McNerney, Ruth; Mallard, Kim; Hill-Cawthorne, Grant; Coll, Francesc; Nair, Mridul; Pain, Arnab; Clark, Taane G; Hasan, Rumina

2015-01-01

Improved molecular diagnostic methods for detection drug resistance in Mycobacterium tuberculosis (MTB) strains are required. Resistance to first- and second- line anti-tuberculous drugs has been associated with single nucleotide polymorphisms (SNPs) in particular genes. However, these SNPs can vary between MTB lineages therefore local data is required to describe different strain populations. We used whole genome sequencing (WGS) to characterize 37 extensively drug-resistant (XDR) MTB isolates from Pakistan and investigated 40 genes associated with drug resistance. Rifampicin resistance was attributable to SNPs in the rpoB hot-spot region. Isoniazid resistance was most commonly associated with the katG codon 315 (92%) mutation followed by inhA S94A (8%) however, one strain did not have SNPs in katG, inhA or oxyR-ahpC. All strains were pyrazimamide resistant but only 43% had pncA SNPs. Ethambutol resistant strains predominantly had embB codon 306 (62%) mutations, but additional SNPs at embB codons 406, 378 and 328 were also present. Fluoroquinolone resistance was associated with gyrA 91-94 codons in 81% of strains; four strains had only gyrB mutations, while others did not have SNPs in either gyrA or gyrB. Streptomycin resistant strains had mutations in ribosomal RNA genes; rpsL codon 43 (42%); rrs 500 region (16%), and gidB (34%) while six strains did not have mutations in any of these genes. Amikacin/kanamycin/capreomycin resistance was associated with SNPs in rrs at nt1401 (78%) and nt1484 (3%), except in seven (19%) strains. We estimate that if only the common hot-spot region targets of current commercial assays were used, the concordance between phenotypic and genotypic testing for these XDR strains would vary between rifampicin (100%), isoniazid (92%), flouroquinolones (81%), aminoglycoside (78%) and ethambutol (62%); while pncA sequencing would provide genotypic resistance in less than half the isolates. This work highlights the importance of expanded targets for drug resistance detection in MTB isolates.

Caledonian scots pine: origins and genetic structure

Treesearch

Bohun B Kinloch; R. D. Westfall; G. I. Forrest

1986-01-01

Monoterpene and isozyme loci, used as markers to study the genetic structure of Scots pine (Pinus sylvestris L.) native to Scotland, showed that the endemic populations are not genetically impoverished, in spite of severe contraction in range and numbers as a result of both natural and anthropogenic causes. On the contrary, variability in the relict...

Scottish Classroom Voices: A Case Study of Teaching and Learning Scots

ERIC Educational Resources Information Center

Shoba, Jo Arthur

2010-01-01

Research in multilingual classrooms demonstrates education as a key site within which social and linguistic values are shaped. This study extends such research by investigating language use in a Scottish primary classroom. Scots is widely spoken throughout Scotland, figuring in a 2003 Scottish Parliament report as one of two indigenous heritage…

Delivering Sustainable Practice? A Case Study of the Scottish Active Schools Programme

ERIC Educational Resources Information Center

Reid, Gavin

2009-01-01

Since 1999, concerns about Scotland's future health and economic performance have profoundly impacted on the new Scottish Executive. Research highlighting an obesity crisis facing young Scots has, together with the work of Scotland's Physical Activity Task Force and Physical Education Review Group, encouraged the education of all young Scots to be…

Ectomycorrhizae of young and mature Scots pine trees in industrial regions in Poland

Treesearch

Barbara Kieliszewska-Rokicka; Maria Rudawska; Tomasz Leski

1998-01-01

Ectomycorrhizae of Scots pine (Pinus sylvestris L.) trees grown in forests influenced by different levels of air pollutants were investigated. Total numbers of mycorrhizal root tips in the soil horizons and the frequency of mycorrhizal morphotypes were compared as indicators of ectomycorrhizal status. The studies were conducted in two comparable...

Stem Cell Ophthalmology Treatment Study (SCOTS): bone marrow-derived stem cells in the treatment of Leber's hereditary optic neuropathy

PubMed Central

Weiss, Jeffrey N.; Levy, Steven; Benes, Susan C.

2016-01-01

The Stem Cell Ophthalmology Treatment Study (SCOTS) is currently the largest-scale stem cell ophthalmology trial registered at ClinicalTrials.gov (identifier: NCT01920867). SCOTS utilizes autologous bone marrow-derived stem cells (BMSCs) to treat optic nerve and retinal diseases. Treatment approaches include a combination of retrobulbar, subtenon, intravitreal, intra-optic nerve, subretinal, and intravenous injection of autologous BMSCs according to the nature of the disease, the degree of visual loss, and any risk factors related to the treatments. Patients with Leber's hereditary optic neuropathy had visual acuity gains on the Early Treatment Diabetic Retinopathy Study (ETDRS) of up to 35 letters and Snellen acuity improvements from hand motion to 20/200 and from counting fingers to 20/100. Visual field improvements were noted. Macular and optic nerve head nerve fiber layer typically thickened. No serious complications were seen. The increases in visual acuity obtained in our study were encouraging and suggest that the use of autologous BMSCs as provided in SCOTS for ophthalmologic mitochondrial diseases including Leber's hereditary optic neuropathy may be a viable treatment option. PMID:27904503

Contribution of single amino acid and codon substitutions to the production and secretion of a lipase by Bacillus subtilis.

PubMed

Skoczinski, Pia; Volkenborn, Kristina; Fulton, Alexander; Bhadauriya, Anuseema; Nutschel, Christina; Gohlke, Holger; Knapp, Andreas; Jaeger, Karl-Erich

2017-09-25

Bacillus subtilis produces and secretes proteins in amounts of up to 20 g/l under optimal conditions. However, protein production can be challenging if transcription and cotranslational secretion are negatively affected, or the target protein is degraded by extracellular proteases. This study aims at elucidating the influence of a target protein on its own production by a systematic mutational analysis of the homologous B. subtilis model protein lipase A (LipA). We have covered the full natural diversity of single amino acid substitutions at 155 positions of LipA by site saturation mutagenesis excluding only highly conserved residues and qualitatively and quantitatively screened about 30,000 clones for extracellular LipA production. Identified variants with beneficial effects on production were sequenced and analyzed regarding B. subtilis growth behavior, extracellular lipase activity and amount as well as changes in lipase transcript levels. In total, 26 LipA variants were identified showing an up to twofold increase in either amount or activity of extracellular lipase. These variants harbor single amino acid or codon substitutions that did not substantially affect B. subtilis growth. Subsequent exemplary combination of beneficial single amino acid substitutions revealed an additive effect solely at the level of extracellular lipase amount; however, lipase amount and activity could not be increased simultaneously. Single amino acid and codon substitutions can affect LipA secretion and production by B. subtilis. Several codon-related effects were observed that either enhance lipA transcription or promote a more efficient folding of LipA. Single amino acid substitutions could improve LipA production by increasing its secretion or stability in the culture supernatant. Our findings indicate that optimization of the expression system is not sufficient for efficient protein production in B. subtilis. The sequence of the target protein should also be considered as an optimization target for successful protein production. Our results further suggest that variants with improved properties might be identified much faster and easier if mutagenesis is prioritized towards elements that contribute to enzymatic activity or structural integrity.

Drought stress alters the concentration of wood terpenoids in Scots pine and Norway spruce seedlings.

PubMed

Turtola, Satu; Manninen, Anne-Marja; Rikala, Risto; Kainulainen, Pirjo

2003-09-01

Drought is known to have an impact on the resistance of conifers to various pests, for example, by affecting resin flow in trees. Little is known, however, about the quantitative and qualitative changes in resin when trees are growing in low moisture conditions. We exposed Scots pine (Pinus sylvestris L.) and Norway spruce (Picea abies (L.) Karst.) seedlings to medium and severe drought stress for two growing seasons and analyzed the monoterpenes and resin acids in the main stem wood after two years of treatment. In addition to secondary chemistry, we measured the level of nutrients in the needles and the growth response of seedlings. After the first year of treatment, drought stress did not affect the growth of seedlings, but in the second year, shoot growth was retarded, especially in Scots pine. In both conifer species, severe drought increased the concentrations of several individual monoterpenes and resin acids. Total monoterpenes and resin acids were 39 and 32% higher in severe drought-treated Scots pine seedlings than in the controls, and 35 and 45% higher in Norway spruce seedlings. In Scots pine needles, the concentrations of nitrogen and phosphorus increased, while magnesium and calcium decreased compared to controls. In Norway spruce needles, nutrient concentrations were not affected. The results suggest that drought stress substantially affects both the growth of conifers and the chemical quality of the wood. We discuss the potential trade-off in growth and defense of small conifer seedlings.

A Stem-Loop Structure in Potato Leafroll Virus Open Reading Frame 5 (ORF5) Is Essential for Readthrough Translation of the Coat Protein ORF Stop Codon 700 Bases Upstream.

PubMed

Xu, Yi; Ju, Ho-Jong; DeBlasio, Stacy; Carino, Elizabeth J; Johnson, Richard; MacCoss, Michael J; Heck, Michelle; Miller, W Allen; Gray, Stewart M

2018-06-01

Translational readthrough of the stop codon of the capsid protein (CP) open reading frame (ORF) is used by members of the Luteoviridae to produce their minor capsid protein as a readthrough protein (RTP). The elements regulating RTP expression are not well understood, but they involve long-distance interactions between RNA domains. Using high-resolution mass spectrometry, glutamine and tyrosine were identified as the primary amino acids inserted at the stop codon of Potato leafroll virus (PLRV) CP ORF. We characterized the contributions of a cytidine-rich domain immediately downstream and a branched stem-loop structure 600 to 700 nucleotides downstream of the CP stop codon. Mutations predicted to disrupt and restore the base of the distal stem-loop structure prevented and restored stop codon readthrough. Motifs in the downstream readthrough element (DRTE) are predicted to base pair to a site within 27 nucleotides (nt) of the CP ORF stop codon. Consistent with a requirement for this base pairing, the DRTE of Cereal yellow dwarf virus was not compatible with the stop codon-proximal element of PLRV in facilitating readthrough. Moreover, deletion of the complementary tract of bases from the stop codon-proximal region or the DRTE of PLRV prevented readthrough. In contrast, the distance and sequence composition between the two domains was flexible. Mutants deficient in RTP translation moved long distances in plants, but fewer infection foci developed in systemically infected leaves. Selective 2'-hydroxyl acylation and primer extension (SHAPE) probing to determine the secondary structure of the mutant DRTEs revealed that the functional mutants were more likely to have bases accessible for long-distance base pairing than the nonfunctional mutants. This study reveals a heretofore unknown combination of RNA structure and sequence that reduces stop codon efficiency, allowing translation of a key viral protein. IMPORTANCE Programmed stop codon readthrough is used by many animal and plant viruses to produce key viral proteins. Moreover, such "leaky" stop codons are used in host mRNAs or can arise from mutations that cause genetic disease. Thus, it is important to understand the mechanism(s) of stop codon readthrough. Here, we shed light on the mechanism of readthrough of the stop codon of the coat protein ORFs of viruses in the Luteoviridae by identifying the amino acids inserted at the stop codon and RNA structures that facilitate this "leakiness" of the stop codon. Members of the Luteoviridae encode a C-terminal extension to the capsid protein known as the readthrough protein (RTP). We characterized two RNA domains in Potato leafroll virus (PLRV), located 600 to 700 nucleotides apart, that are essential for efficient RTP translation. We further determined that the PLRV readthrough process involves both local structures and long-range RNA-RNA interactions. Genetic manipulation of the RNA structure altered the ability of PLRV to translate RTP and systemically infect the plant. This demonstrates that plant virus RNA contains multiple layers of information beyond the primary sequence and extends our understanding of stop codon readthrough. Strategic targets that can be exploited to disrupt the virus life cycle and reduce its ability to move within and between plant hosts were revealed. Copyright © 2018 American Society for Microbiology.

Know your limits? Climate extremes impact the range of Scots pine in unexpected places

PubMed Central

Julio Camarero, J.; Gazol, Antonio; Sancho-Benages, Santiago; Sangüesa-Barreda, Gabriel

2015-01-01

Background and Aims Although extreme climatic events such as drought are known to modify forest dynamics by triggering tree dieback, the impact of extreme cold events, especially at the low-latitude margin (‘rear edge’) of species distributional ranges, has received little attention. The aim of this study was to examine the impact of one such extreme cold event on a population of Scots pine (Pinus sylvestris) along the species’ European southern rear-edge range limit and to determine how such events can be incorporated into species distribution models (SDMs). Methods A combination of dendrochronology and field observation was used to quantify how an extreme cold event in 2001 in eastern Spain affected growth, needle loss and mortality of Scots pine. Long-term European climatic data sets were used to contextualize the severity of the 2001 event, and an SDM for Scots pine in Europe was used to predict climatic range limits. Key Results The 2001 winter reached record minimum temperatures (equivalent to the maximum European-wide diurnal ranges) and, for trees already stressed by a preceding dry summer and autumn, this caused dieback and large-scale mortality. Needle loss and mortality were particularly evident in south-facing sites, where post-event recovery was greatly reduced. The SDM predicted European Scots pine distribution mainly on the basis of responses to maximum and minimum monthly temperatures, but in comparison with this the observed effects of the 2001 cold event at the southerly edge of the range limit were unforeseen. Conclusions The results suggest that in order to better forecast how anthropogenic climate change might affect future forest distributions, distribution modelling techniques such as SDMs must incorporate climatic extremes. For Scots pine, this study shows that the effects of cold extremes should be included across the entire distribution margin, including the southern ‘rear edge’, in order to avoid biased predictions based solely on warmer climatic scenarios. PMID:26292992

Contrasting Hydraulic Architectures of Scots Pine and Sessile Oak at Their Southernmost Distribution Limits

PubMed Central

Martínez-Sancho, Elisabet; Dorado-Liñán, Isabel; Hacke, Uwe G.; Seidel, Hannes; Menzel, Annette

2017-01-01

Many temperate European tree species have their southernmost distribution limits in the Mediterranean Basin. The projected climatic conditions, particularly an increase in dryness, might induce an altitudinal and latitudinal retreat at their southernmost distribution limit. Therefore, characterizing the morphological and physiological variability of temperate tree species under dry conditions is essential to understand species’ responses to expected climate change. In this study, we compared branch-level hydraulic traits of four Scots pine and four sessile oak natural stands located at the western and central Mediterranean Basin to assess their adjustment to water limiting conditions. Hydraulic traits such as xylem- and leaf-specific maximum hydraulic conductivity (KS-MAX and KL-MAX), leaf-to-xylem area ratio (AL:AX) and functional xylem fraction (FX) were measured in July 2015 during a long and exceptionally dry summer. Additionally, xylem-specific native hydraulic conductivity (KS-N) and native percentage of loss of hydraulic conductivity (PLC) were measured for Scots pine. Interspecific differences in these hydraulic traits as well as intraspecific variability between sites were assessed. The influence of annual, summer and growing season site climatic aridity (P/PET) on intraspecific variability was investigated. Sessile oak displayed higher values of KS-MAX, KL-MAX, AL:AX but a smaller percentage of FX than Scots pines. Scots pine did not vary in any of the measured hydraulic traits across the sites, and PLC values were low for all sites, even during one of the warmest summers in the region. In contrast, sessile oak showed significant differences in KS-MAX, KL-MAX, and FX across sites, which were significantly related to site aridity. The striking similarity in the hydraulic traits across Scots pine sites suggests that no adjustment in hydraulic architecture was needed, likely as a consequence of a drought-avoidance strategy. In contrast, sessile oak displayed adjustments in the hydraulic architecture along an aridity gradient, pointing to a drought-tolerance strategy. PMID:28473841

Sense codon emancipation for proteome-wide incorporation of noncanonical amino acids: rare isoleucine codon AUA as a target for genetic code expansion

PubMed Central

Bohlke, Nina; Budisa, Nediljko

2014-01-01

One of the major challenges in contemporary synthetic biology is to find a route to engineer synthetic organisms with altered chemical constitution. In terms of core reaction types, nature uses an astonishingly limited repertoire of chemistries when compared with the exceptionally rich and diverse methods of organic chemistry. In this context, the most promising route to change and expand the fundamental chemistry of life is the inclusion of amino acid building blocks beyond the canonical 20 (i.e. expanding the genetic code). This strategy would allow the transfer of numerous chemical functionalities and reactions from the synthetic laboratory into the cellular environment. Due to limitations in terms of both efficiency and practical applicability, state-of-the-art nonsense suppression- or frameshift suppression-based methods are less suitable for such engineering. Consequently, we set out to achieve this goal by sense codon emancipation, that is, liberation from its natural decoding function – a prerequisite for the reassignment of degenerate sense codons to a new 21st amino acid. We have achieved this by redesigning of several features of the post-transcriptional modification machinery which are directly involved in the decoding process. In particular, we report first steps towards the reassignment of 5797 AUA isoleucine codons in Escherichia coli using efficient tools for tRNA nucleotide modification pathway engineering. PMID:24433543

Strange, incredible and impossible things: the early anthropology of Reginald Scot.

PubMed

Littlewood, Roland

2009-06-01

Reginald Scot has been acclaimed as an early rationalist for his critical consideration of witchcraft in 1584. At the same time, the Discoverie of Witchcraft appears organized much as later classic anthropological monographs. This article considers whether his methods and writing might indeed correspond to what we recognise as the procedures of medical or psychiatric anthropology.

Research on Building Education & Workforce Capacity in Systems Engineering

DTIC Science & Technology

2012-09-30

Science Coast Guard Academy Chris Lund, Research Engineer USCG R&D center Civil Engineering Coast Guard Academy Scot T. Tripp, Program Manager USCG...74 researchers Coast Guard Academy Scot T. Tripp, Program Manager Internal institutional USCG R&D center... Woods Industry Lockheed Martin Aeronautics Company Defense contracted system development and analysis Stevens Tom Newby Industry Buro

Scots pine in eastern Nebraska: A provenance study

Treesearch

Ralph A. Read

1971-01-01

Seedling progenies of 36 rangewide provenances of Scots pine (Pinus sylvestris) were established in a field test in eastern Nebraska. Results in growth and other characteristics after 8 years reveal that (1) southern origins bordering the Mediterranean grow slowly to moderately fast and remain dark green in winter, (2) central European origins grow very fast and turn...

Tomicus piniperda (Coleoptera: Scolytidae) reproduction and development in Scots, jack, red and eastern white pine under laboratory conditions

Treesearch

Hui Ye; Robert A. Haack; Toby R. Petrice

2005-01-01

The pine shoot beetle, Tomicus piniperda (L.) (Coleoptera: Scolytidae), is an exotic bark beetle in North America that was first found in the Great Lakes region in 1992. We evaluated T. piniperda reproduction and development in one Eurasian pine (Scots pine, Pinus sylvestris L.) and three North American...

Detection of EGFR Gene Mutation by Mutation-oriented LAMP Method.

PubMed

Matsumoto, Naoyuki; Kumasaka, Akira; Ando, Tomohiro; Komiyama, Kazuo

2018-04-01

Epidermal growth factor receptor (EGFR) is a target of molecular therapeutics for non-small cell lung cancer. EGFR gene mutations at codons 746-753 promote constitutive EGFR activation and result in worst prognosis. However, these mutations augment the therapeutic effect of EGFR-tyrosine kinase inhibitor. Therefore, the detection of EGFR gene mutations is important for determining treatment planning. The aim of the study was to establish a method to detect EGFR gene mutations at codons 746-753. EGFR gene mutation at codons 746-753 in six cancer cell lines were investigated. A loop-mediated isothermal amplification (LAMP)-based procedure was developed, that employed peptide nucleic acid to suppress amplification of the wild-type allele. This mutation-oriented LAMP can amplify the DNA fragment of the EGFR gene with codons 746-753 mutations within 30 min. Moreover, boiled cells can work as template resources. Mutation oriented-LAMP assay for EGFR gene mutation is sensitive on extracted DNA. This procedure would be capable of detecting EGFR gene mutation in sputum, pleural effusion, broncho-alveolar lavage fluid or trans-bronchial lung biopsy by chair side. Copyright© 2018, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.

Effectiveness of newspaper advertising for patient recruitment into a clinical trial.

PubMed

Hapca, Adrian; Jennings, Claudine G; Wei, Li; Wilson, Adam; MacDonald, Thomas M; Mackenzie, Isla S

2014-06-01

To measure the impact of newspaper advertising across Scotland on patient interest, and subsequent recruitment into the Standard Care vs. Celecoxib Outcome Trial (SCOT), a clinical trial investigating the cardiovascular safety of non-steroidal anti-inflammatory drugs in patients with osteoarthritis or rheumatoid arthritis. Newspaper advertisements about the SCOT trial were placed sequentially in regional and national Scottish newspapers. The number of phone calls as a result of exposure to the advertisements and ongoing study recruitment rates were recorded before, during and after the advertising campaign. To enroll in SCOT individuals had to be registered with a participating GP practice. The total cost for the advertising campaign was £46 250 and 320 phone calls were received as a result of individuals responding to the newspaper advertisements. One hundred and seventy-two individuals were identified as possibly suitable to be included in the study. However only 36 were registered at participating GP practices, 17 completed a screening visit and 15 finally were randomized into the study. The average cost per respondent individual was £144 and the average cost per randomized patient was £3083. Analysis of recruitment rate trends showed that there was no impact of the newspaper advertising campaign on increasing recruitment into SCOT. Advertisements placed in local and national newspapers were not an effective recruitment strategy for the SCOT trial. The advertisements attracted relatively small numbers of respondents, many of whom did not meet study inclusion criteria or were not registered at a participating GP practice. © 2013 The British Pharmacological Society.

Features of Scots pine radial growth in conditions of provenance trial

NASA Astrophysics Data System (ADS)

Kuzmin, S.

2012-12-01

Provenance trial of Scots pine in Boguchany forestry of Krasnoyarsk krai is conducted on two different soils - dark-grey loam forest soil and sod-podzol sandy soil. Complex of negative factors for plant growth and development appears in dry conditions of sandy soil. It could results in decrease of resistance to diseases. Sandy soils in different climatic zones have such common traits as low absorbing capacity, poorness of elemental nutrition, low microbiological activity and moisture capacity, very high water permeability. But Scots pine trees growing in such conditions could have certain advantages and perspectives of use. In the scope of climate change (global warming) the study of Scots pine growth on sandy soil become urgent because of more frequent appearance of dry seasons. Purpose of the work is revelation of radial growth features of Scots pine with different origin in dry conditions of sandy soil and assessment of external factors influence. The main feature of radial growth of majority of studied pine provenances in conditions of sandy soil is presence of significant variation of increment with distinct decline in 25-years old with loss of tree rings in a number of cases. The reason of it is complex of factors: deficit of June precipitation and next following outbreak of fungal disease. Found «frost rings» for all trees of studied clymatypes in 1992 are the consequence of temperature decline from May 21 to June 2 - from 23 C degrees up to 2 C. Perspective climatypes with biggest radial increments and least sensitivity to fungal disease were revealed.

Effectiveness of newspaper advertising for patient recruitment into a clinical trial

PubMed Central

Hapca, Adrian; Jennings, Claudine G; Wei, Li; Wilson, Adam; MacDonald, Thomas M; Mackenzie, Isla S

2014-01-01

Aims To measure the impact of newspaper advertising across Scotland on patient interest, and subsequent recruitment into the Standard Care vs. Celecoxib Outcome Trial (SCOT), a clinical trial investigating the cardiovascular safety of non-steroidal anti-inflammatory drugs in patients with osteoarthritis or rheumatoid arthritis. Methods Newspaper advertisements about the SCOT trial were placed sequentially in regional and national Scottish newspapers. The number of phone calls as a result of exposure to the advertisements and ongoing study recruitment rates were recorded before, during and after the advertising campaign. To enroll in SCOT individuals had to be registered with a participating GP practice. Results The total cost for the advertising campaign was £46 250 and 320 phone calls were received as a result of individuals responding to the newspaper advertisements. One hundred and seventy-two individuals were identified as possibly suitable to be included in the study. However only 36 were registered at participating GP practices, 17 completed a screening visit and 15 finally were randomized into the study. The average cost per respondent individual was £144 and the average cost per randomized patient was £3083. Analysis of recruitment rate trends showed that there was no impact of the newspaper advertising campaign on increasing recruitment into SCOT. Conclusions Advertisements placed in local and national newspapers were not an effective recruitment strategy for the SCOT trial. The advertisements attracted relatively small numbers of respondents, many of whom did not meet study inclusion criteria or were not registered at a participating GP practice. PMID:24283948

Additive Manufacturing: Which DLA-Managed Legacy Parts are Potential AM Candidates

DTIC Science & Technology

2016-07-01

william.t.veney.civ@mail.mil Mr. Scot Seitz Army/G4 717-770-4304 scot.s.seitz.civ@mail.mil Mr. Stephen Luckowski Army/RDECOM 973-724-3100...stephen.l.luckowski.civ@mail.mil Maj. Eric Kirchner Marine Corps/I&L 571-256-7105 eric.kirchner@usmc.mil Capt. Christopher Wood Marine Corps/I&L 571-256-2740

Scots and Scotch Irish in Connecticut: A History. The Peoples of Connecticut Multicultural Ethnic Heritage Project, Number Five.

ERIC Educational Resources Information Center

Stone, Frank Andrews

This monograph reviews the history of Scots and Scotch Irish settlements in Connecticut from the 17th century to the present. Intended primarily for adult readers, the pamphlet is part of a series of curriculum guides about ethnic groups in America. Historical information was obtained from oral histories, church and military records, and…

Rhesus Monkey Rhadinovirus ORF57 Induces gH and gL Glycoprotein Expression through Posttranscriptional Accumulation of Target mRNAs ▿

PubMed Central

Shin, Young C.; Desrosiers, Ronald C.

2011-01-01

Open reading frame 57 (ORF57) of gamma-2 herpesviruses is a key regulator of viral gene expression. It has been reported to enhance the expression of viral genes by transcriptional, posttranscriptional, or translational activation mechanisms. Previously we have shown that the expression of gH and gL of rhesus monkey rhadinovirus (RRV), a close relative of the human Kaposi's sarcoma-associated herpesvirus (KSHV), could be dramatically rescued by codon optimization as well as by ORF57 coexpression (J. P. Bilello, J. S. Morgan, and R. C. Desrosiers, J. Virol. 82:7231–7237, 2008). We show here that ORF57 coexpression and codon optimization had similar effects, except that the rescue of expression by codon optimization was temporally delayed relative to that of ORF57 coexpression. The transfection of gL mRNA directly into cells with or without ORF57 coexpression and with or without codon optimization recapitulated the effects of these modes of induction on transfected DNA. These findings suggested an important role for the enhancement of mRNA stability and/or the translation of mRNA for these very different modes of induced expression. This conclusion was confirmed by several different measures of gH and gL mRNA stability and accumulation with or without ORF57 coexpression and with or without codon optimization. Our results indicate that RRV gH and gL expression is severely limited by the stability of the mRNA and that ORF57 coexpression and codon optimization independently induce gH and gL expression principally by allowing accumulation and translation of these mRNAs. PMID:21613403

Is Mutation Random or Targeted?: No Evidence for Hypermutability in Snail Toxin Genes.

PubMed

Roy, Scott W

2016-10-01

Ever since Luria and Delbruck, the notion that mutation is random with respect to fitness has been foundational to modern biology. However, various studies have claimed striking exceptions to this rule. One influential case involves toxin-encoding genes in snails of the genus Conus, termed conotoxins, a large gene family that undergoes rapid diversification of their protein-coding sequences by positive selection. Previous reconstructions of the sequence evolution of conotoxin genes claimed striking patterns: (1) elevated synonymous change, interpreted as being due to targeted "hypermutation" in this region; (2) elevated transversion-to-transition ratios, interpreted as reflective of the particular mechanism of hypermutation; and (3) much lower rates of synonymous change in the codons encoding several highly conserved cysteine residues, interpreted as strong position-specific codon bias. This work has spawned a variety of studies on the potential mechanisms of hypermutation and on causes for cysteine codon bias, and has inspired hypermutation hypotheses for various other fast-evolving genes. Here, I show that all three findings are likely to be artifacts of statistical reconstruction. First, by simulating nonsynonymous change I show that high rates of dN can lead to overestimation of dS. Second, I show that there is no evidence for any of these three patterns in comparisons of closely related conotoxin sequences, suggesting that the reported findings are due to breakdown of statistical methods at high levels of sequence divergence. The current findings suggest that mutation and codon bias in conotoxin genes may not be atypical, and that random mutation and selection can explain the evolution of even these exceptional loci. © The Author 2016. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

mRNA 3' of the A site bound codon is located close to protein S3 on the human 80S ribosome.

PubMed

Molotkov, Maxim V; Graifer, Dmitri M; Popugaeva, Elena A; Bulygin, Konstantin N; Meschaninova, Maria I; Ven'yaminova, Aliya G; Karpova, Galina G

2006-07-01

Ribosomal proteins neighboring the mRNA downstream of the codon bound at the decoding site of human 80S ribosomes were identified using three sets of mRNA analogues that contained a UUU triplet at the 5' terminus and a perfluorophenylazide cross-linker at guanosine, adenosine or uridine residues placed at various locations 3' of this triplet. The positions of modified mRNA nucleotides on the ribosome were governed by tRNA(Phe) cognate to the UUU triplet targeted to the P site. Upon mild UV-irradiation, the mRNA analogues cross-linked preferentially to the 40S subunit, to the proteins and to a lesser extent to the 18S rRNA. Cross-linked nucleotides of 18S rRNA were identified previously. In the present study, it is shown that among the proteins the main target for cross-linking with all the mRNA analogues tested was protein S3 (homologous to prokaryotic S3, S3p); minor cross-linking to protein S2 (S5p) was also detected. Both proteins cross-linked to mRNA analogues in the ternary complexes as well as in the binary complexes (without tRNA). In the ternary complexes protein S15 (S19p) also cross-linked, the yield of the cross-link decreased significantly when the modified nucleotide moved from position +5 to position +12 with respect to the first nucleotide of the P site bound codon. In several ternary complexes minor cross-linking to protein S30 was likewise detected. The results of this study indicate that S3 is a key protein at the mRNA binding site neighboring mRNA downstream of the codon at the decoding site in the human ribosome.

Sense codon emancipation for proteome-wide incorporation of noncanonical amino acids: rare isoleucine codon AUA as a target for genetic code expansion.

PubMed

Bohlke, Nina; Budisa, Nediljko

2014-02-01

One of the major challenges in contemporary synthetic biology is to find a route to engineer synthetic organisms with altered chemical constitution. In terms of core reaction types, nature uses an astonishingly limited repertoire of chemistries when compared with the exceptionally rich and diverse methods of organic chemistry. In this context, the most promising route to change and expand the fundamental chemistry of life is the inclusion of amino acid building blocks beyond the canonical 20 (i.e. expanding the genetic code). This strategy would allow the transfer of numerous chemical functionalities and reactions from the synthetic laboratory into the cellular environment. Due to limitations in terms of both efficiency and practical applicability, state-of-the-art nonsense suppression- or frameshift suppression-based methods are less suitable for such engineering. Consequently, we set out to achieve this goal by sense codon emancipation, that is, liberation from its natural decoding function - a prerequisite for the reassignment of degenerate sense codons to a new 21st amino acid. We have achieved this by redesigning of several features of the post-transcriptional modification machinery which are directly involved in the decoding process. In particular, we report first steps towards the reassignment of 5797 AUA isoleucine codons in Escherichia coli using efficient tools for tRNA nucleotide modification pathway engineering. © 2014 The Authors. FEMS Microbiology Letters published by John Wiley & Sons Ltd on behalf of the Federation of European Microbiological Societies.

Translation, modification and cellular distribution of two AC4 variants of African cassava mosaic virus in yeast and their pathogenic potential in plants

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

Hipp, Katharina, E-mail: katharina.hipp@bio.uni-st

Plant infecting geminiviruses encode a small (A)C4 protein within the open reading frame of the replication-initiator protein. In African cassava mosaic virus, two in-frame start codons may be used for the translation of a longer and a shorter AC4 variant. Both were fused to green fluorescent protein or glutathione-S-transferase genes and expressed in fission yeast. The longer variant accumulated in discrete spots in the cytoplasm, whereas the shorter variant localized to the plasma membrane. A similar expression pattern was found in plants. A myristoylation motif may promote a targeting of the shorter variant to the plasma membrane. Mass spectrometry analysismore » of the yeast-expressed shorter variant detected the corresponding myristoylation. The biological relevance of the second start codon was confirmed using mutated infectious clones. Whereas mutating the first start codon had no effect on the infectivity in Nicotiana benthamiana plants, the second start codon proved to be essential. -- Highlights: •The ACMV AC4 may be translated from one or the other in-frame start codon. •Both AC4 variants are translated in fission yeast. •The long AC4 protein localizes to the cytoplasm, the short to the plasma membrane. •The short variant is myristoylated in yeast and may promote membrane localization. •Only the shorter AC4 variant has an impact on viral infections in plants.« less

Peroxisomal lactate dehydrogenase is generated by translational readthrough in mammals

PubMed Central

Schueren, Fabian; Lingner, Thomas; George, Rosemol; Hofhuis, Julia; Dickel, Corinna; Gärtner, Jutta; Thoms, Sven

2014-01-01

Translational readthrough gives rise to low abundance proteins with C-terminal extensions beyond the stop codon. To identify functional translational readthrough, we estimated the readthrough propensity (RTP) of all stop codon contexts of the human genome by a new regression model in silico, identified a nucleotide consensus motif for high RTP by using this model, and analyzed all readthrough extensions in silico with a new predictor for peroxisomal targeting signal type 1 (PTS1). Lactate dehydrogenase B (LDHB) showed the highest combined RTP and PTS1 probability. Experimentally we show that at least 1.6% of the total cellular LDHB is targeted to the peroxisome by a conserved hidden PTS1. The readthrough-extended lactate dehydrogenase subunit LDHBx can also co-import LDHA, the other LDH subunit, into peroxisomes. Peroxisomal LDH is conserved in mammals and likely contributes to redox equivalent regeneration in peroxisomes. DOI: http://dx.doi.org/10.7554/eLife.03640.001 PMID:25247702

The Genesis of Transformation: The Rise of the United States Army’s Modular Brigade Combat Teams

DTIC Science & Technology

2013-03-01

transportation or employment. “Organized around dismounted infantry,” IBCT’s were “optimized for operations in close terrain, such as swamps, woods ...requirements.”132 While many of these 129 John Matsumura, Randall Steeb, Thomas Herbert, Scot ...War. Cambridge: Harvard University Press, 2007. Matsumura, John, Randall Steeb, Thomas Herbert, Scot Eisenhard, John Gordon, Mark Lees, and Gail

Chemical composition of needles and cambial activity of stems of Scots pine trees affected by air pollutants in Polish forests

Treesearch

Wojciech Dmuchowski; Ewa U. Kurczynska; Wieslaw Wloch

1998-01-01

The impact of environmental pollution is defined for the chemical composition of Scots pine (Pinus sylvestris L.) needles and cambial activity in the tree stems in Polish forests. The research investigated 20-year-old trees growing in two areas in significantly different levels of pollution. The highly polluted area was located near the Warsaw...

Know your limits? Climate extremes impact the range of Scots pine in unexpected places.

PubMed

Julio Camarero, J; Gazol, Antonio; Sancho-Benages, Santiago; Sangüesa-Barreda, Gabriel

2015-11-01

Although extreme climatic events such as drought are known to modify forest dynamics by triggering tree dieback, the impact of extreme cold events, especially at the low-latitude margin ('rear edge') of species distributional ranges, has received little attention. The aim of this study was to examine the impact of one such extreme cold event on a population of Scots pine (Pinus sylvestris) along the species' European southern rear-edge range limit and to determine how such events can be incorporated into species distribution models (SDMs). A combination of dendrochronology and field observation was used to quantify how an extreme cold event in 2001 in eastern Spain affected growth, needle loss and mortality of Scots pine. Long-term European climatic data sets were used to contextualize the severity of the 2001 event, and an SDM for Scots pine in Europe was used to predict climatic range limits. The 2001 winter reached record minimum temperatures (equivalent to the maximum European-wide diurnal ranges) and, for trees already stressed by a preceding dry summer and autumn, this caused dieback and large-scale mortality. Needle loss and mortality were particularly evident in south-facing sites, where post-event recovery was greatly reduced. The SDM predicted European Scots pine distribution mainly on the basis of responses to maximum and minimum monthly temperatures, but in comparison with this the observed effects of the 2001 cold event at the southerly edge of the range limit were unforeseen. The results suggest that in order to better forecast how anthropogenic climate change might affect future forest distributions, distribution modelling techniques such as SDMs must incorporate climatic extremes. For Scots pine, this study shows that the effects of cold extremes should be included across the entire distribution margin, including the southern 'rear edge', in order to avoid biased predictions based solely on warmer climatic scenarios. © The Author 2015. Published by Oxford University Press on behalf of the Annals of Botany Company. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

Predicting the decomposition of Scots pine, Norway spruce, and birch stems in Finland.

PubMed

Mäkinen, Harri; Hynynen, Jari; Siitonen, Juha; Sievänen, Risto

2006-10-01

Models were developed for predicting the decomposition of dead wood for the main tree species in Finland, based on data collected from long-term thinning experiments in southern and central Finland. The decomposition rates were strongly related to the number of years after tree death. In contrast to previous studies, which have used the first-order exponential model, we found that the decomposition rate was not constant. Therefore, the Gompertz and Chapman-Richard's functions were fitted to the data. The slow initial decomposition period was mainly due to the fact that most dead trees remained standing as snags after their death. The initial period was followed by a period of rapid decomposition and, finally, by a period of moderately slow decomposition. Birch stems decomposed more rapidly than Scots pine and Norway spruce stems. Decomposition rates of Norway spruce stems were somewhat lower than those of Scots pine. Because the carbon concentration of decaying boles was relatively stable (about 50%) the rate of carbon loss follows that of mass loss. Models were also developed for the probability that a dead tree remains standing as a snag. During the first years after death, the probability was high. Thereafter, it decreased rapidly, the decrease being faster for birch stems than for Scots pine and Norway spruce stems. Almost all stems had fallen down within 40 years after their death. In Scots pine and Norway spruce, most snags remained hard and belonged to decay class 1. In birch, a higher proportion of snags belonged to the more advanced decay classes. The models provide a framework for predicting dead wood dynamics in managed as well as dense unthinned stands. The models can be incorporated into forest management planning systems, thereby facilitating estimates of carbon dynamics.

Strong resilience of soil respiration components to drought-induced die-off resulting in forest secondary succession.

PubMed

Barba, Josep; Curiel Yuste, Jorge; Poyatos, Rafael; Janssens, Ivan A; Lloret, Francisco

2016-09-01

How forests cope with drought-induced perturbations and how the dependence of soil respiration on environmental and biological drivers is affected in a warming and drying context are becoming key questions. The aims of this study were to determine whether drought-induced die-off and forest succession were reflected in soil respiration and its components and to determine the influence of climate on the soil respiration components. We used the mesh exclusion method to study seasonal variations in soil respiration (R S) and its components: heterotrophic (R H) and autotrophic (R A) [further split into fine root (R R) and mycorrhizal respiration (R M)] in a mixed Mediterranean forest where Scots pine (Pinus sylvestris L.) is undergoing a drought-induced die-off and is being replaced by holm oak (Quercus ilex L.). Drought-induced pine die-off was not reflected in R S nor in its components, which denotes a high functional resilience of the plant and soil system to pine die-off. However, the succession from Scots pine to holm oak resulted in a reduction of R H and thus in an important decrease of total respiration (R S was 36 % lower in holm oaks than in non-defoliated pines). Furthermore, R S and all its components were strongly regulated by soil water content-and-temperature interaction. Since Scots pine die-off and Quercus species colonization seems to be widely occurring at the driest limit of the Scots pine distribution, the functional resilience of the soil system over die-off and the decrease of R S from Scots pine to holm oak could have direct consequences for the C balance of these ecosystems.

Whole Genome Sequencing Based Characterization of Extensively Drug-Resistant Mycobacterium tuberculosis Isolates from Pakistan

PubMed Central

Ali, Asho; Hasan, Zahra; McNerney, Ruth; Mallard, Kim; Hill-Cawthorne, Grant; Coll, Francesc; Nair, Mridul; Pain, Arnab; Clark, Taane G.; Hasan, Rumina

2015-01-01

Improved molecular diagnostic methods for detection drug resistance in Mycobacterium tuberculosis (MTB) strains are required. Resistance to first- and second- line anti-tuberculous drugs has been associated with single nucleotide polymorphisms (SNPs) in particular genes. However, these SNPs can vary between MTB lineages therefore local data is required to describe different strain populations. We used whole genome sequencing (WGS) to characterize 37 extensively drug-resistant (XDR) MTB isolates from Pakistan and investigated 40 genes associated with drug resistance. Rifampicin resistance was attributable to SNPs in the rpoB hot-spot region. Isoniazid resistance was most commonly associated with the katG codon 315 (92%) mutation followed by inhA S94A (8%) however, one strain did not have SNPs in katG, inhA or oxyR-ahpC. All strains were pyrazimamide resistant but only 43% had pncA SNPs. Ethambutol resistant strains predominantly had embB codon 306 (62%) mutations, but additional SNPs at embB codons 406, 378 and 328 were also present. Fluoroquinolone resistance was associated with gyrA 91–94 codons in 81% of strains; four strains had only gyrB mutations, while others did not have SNPs in either gyrA or gyrB. Streptomycin resistant strains had mutations in ribosomal RNA genes; rpsL codon 43 (42%); rrs 500 region (16%), and gidB (34%) while six strains did not have mutations in any of these genes. Amikacin/kanamycin/capreomycin resistance was associated with SNPs in rrs at nt1401 (78%) and nt1484 (3%), except in seven (19%) strains. We estimate that if only the common hot-spot region targets of current commercial assays were used, the concordance between phenotypic and genotypic testing for these XDR strains would vary between rifampicin (100%), isoniazid (92%), flouroquinolones (81%), aminoglycoside (78%) and ethambutol (62%); while pncA sequencing would provide genotypic resistance in less than half the isolates. This work highlights the importance of expanded targets for drug resistance detection in MTB isolates. PMID:25719196

X-linked nephrogenic diabetes insipidus mutations in North America and the Hopewell hypothesis.

PubMed Central

Bichet, D G; Arthus, M F; Lonergan, M; Hendy, G N; Paradis, A J; Fujiwara, T M; Morgan, K; Gregory, M C; Rosenthal, W; Didwania, A

1993-01-01

In X-linked nephrogenic diabetes insipidus (NDI) the urine of male patients is not concentrated after the administration of the antidiuretic hormone arginine-vasopressin. This disease is due to mutations in the V2 receptor gene that maps to chromosome region Xq28. In 1969, Bode and Crawford suggested that most NDI patients in North America shared common ancestors of Ulster Scot immigrants who arrived in Halifax in 1761 on the ship Hopewell. A link between this family and a large Utah kindred was also suggested. DNA was obtained from 17 affected male patients from the "Hopewell" kindred and from four additional families from Nova Scotia and New Brunswick who shared the same Xq28 NDI haplotype. The Utah kindred and two families (Q2, Q3) from Quebec were also studied. The "Hopewell" mutation, W71X, is a single base substitution (G-->A) that changes codon 71 from TGG (tryptophan) to TGA (stop). The W71X mutation was found in affected members of the Hopewell and of the four satellite families. The W71X mutation is the cause of X-linked NDI for the largest number of related male patients living in North America. Other families (Utah, Q2 and Q3) that are historically and ethnically unrelated bear other mutations in the V2 receptor gene. Images PMID:8104196

The effects of heat treatment on some technological properties of Scots pine (Pinus sylvestris L.) wood.

PubMed

Korkut, Süleyman; Akgül, Mehmet; Dündar, Turker

2008-04-01

Heat treatment is often applied to wood species to improve their dimensional stability. This study examined the effect of heat treatment on certain mechanical properties of Scots pine (Pinus sylvestris L.), which has industrially high usage potential and large plantations in Turkey. Wood specimens obtained from Bolu, Turkey, were subjected to heat treatment under atmospheric pressure at varying temperatures (120, 150 and 180 degrees C) for varying durations (2, 6 and 10h). The test results of heat-treated Scots pine and control samples showed that technological properties including compression strength, bending strength, modulus of elasticity in bending, janka-hardness, impact bending strength and tension strength perpendicular to grain suffered with heat treatment, and increase in temperature and duration further diminished technological strength values of the wood specimens.

Genetic variation, population structure and linkage disequilibrium in Switchgrass with ISSR, SCoT and EST-SSR markers.

PubMed

Zhang, Yu; Yan, Haidong; Jiang, Xiaomei; Wang, Xiaoli; Huang, Linkai; Xu, Bin; Zhang, Xinquan; Zhang, Lexin

2016-01-01

To evaluate genetic variation, population structure, and the extent of linkage disequilibrium (LD), 134 switchgrass ( Panicum virgatum L.) samples were analyzed with 51 markers, including 16 ISSRs, 20 SCoTs, and 15 EST-SSRs. In this study, a high level of genetic variation was observed in the switchgrass samples and they had an average Nei's gene diversity index (H) of 0.311. A total of 793 bands were obtained, of which 708 (89.28 %) were polymorphic. Using a parameter marker index (MI), the efficiency of the three types of markers (ISSR, SCoT, and EST-SSR) in the study were compared and we found that SCoT had a higher marker efficiency than the other two markers. The 134 switchgrass samples could be divided into two sub-populations based on STRUCTURE, UPGMA clustering, and principal coordinate analyses (PCA), and upland and lowland ecotypes could be separated by UPGMA clustering and PCA analyses. Linkage disequilibrium analysis revealed an average r 2 of 0.035 across all 51 markers, indicating a trend of higher LD in sub-population 2 than that in sub-population 1 ( P  < 0.01). The population structure revealed in this study will guide the design of future association studies using these switchgrass samples.

Effects of radioactive contamination on Scots pines in the remote period after the Chernobyl accident.

PubMed

Geras'kin, Stanislav; Oudalova, Alla; Dikareva, Nina; Spiridonov, Sergey; Hinton, Thomas; Chernonog, Elena; Garnier-Laplace, Jacqueline

2011-08-01

A 6 year study of Scots pine populations inhabiting sites in the Bryansk region of Russia radioactively contaminated as a result of the Chernobyl accident is presented. In six study sites, (137)Cs activity concentrations and heavy metal content in soils, as well as (137)Cs, (90)Sr and heavy metal concentrations in cones were measured. Doses absorbed in reproduction organs of pine trees were calculated using a dosimetric model. The maximum annual dose absorbed at the most contaminated site was about 130 mGy. Occurrence of aberrant cells scored in the root meristem of germinated seeds collected from pine trees growing on radioactively contaminated territories for over 20 years significantly exceeded the reference levels during all 6 years of the study. The data suggest that cytogenetic effects occur in Scots pine populations due to the radioactive contamination. However, no consistent differences in reproductive ability were detected between the impacted and reference populations as measured by the frequency of abortive seeds. Even though the Scots pine populations have occupied radioactively contaminated territories for two decades, there were no clear indications of adaptation to the radiation, when measured by the number of aberrant cells in root meristems of seeds exposed to an additional acute dose of radiation.

Enhanced expression of codon optimized Mycobacterium avium subsp. paratuberculosis antigens in Lactobacillus salivarius.

PubMed

Johnston, Christopher D; Bannantine, John P; Govender, Rodney; Endersen, Lorraine; Pletzer, Daniel; Weingart, Helge; Coffey, Aidan; O'Mahony, Jim; Sleator, Roy D

2014-01-01

It is well documented that open reading frames containing high GC content show poor expression in A+T rich hosts. Specifically, G+C-rich codon usage is a limiting factor in heterologous expression of Mycobacterium avium subsp. paratuberculosis (MAP) proteins using Lactobacillus salivarius. However, re-engineering opening reading frames through synonymous substitutions can offset codon bias and greatly enhance MAP protein production in this host. In this report, we demonstrate that codon-usage manipulation of MAP2121c can enhance the heterologous expression of the major membrane protein (MMP), analogous to the form in which it is produced natively by MAP bacilli. When heterologously over-expressed, antigenic determinants were preserved in synthetic MMP proteins as shown by monoclonal antibody mediated ELISA. Moreover, MMP is a membrane protein in MAP, which is also targeted to the cellular surface of recombinant L. salivarius at levels comparable to MAP. Additionally, we previously engineered MAP3733c (encoding MptD) and show herein that MptD displays the tendency to associate with the cytoplasmic membrane boundary under confocal microscopy and the intracellularly accumulated protein selectively adheres to the MptD-specific bacteriophage fMptD. This work demonstrates there is potential for L. salivarius as a viable antigen delivery vehicle for MAP, which may provide an effective mucosal vaccine against Johne's disease.

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

PubMed

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

2007-10-05

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

Genetic diversity and structure of tea plant in Qinba area in China by three types of molecular markers.

PubMed

Zhang, Yu; Zhang, Xiaojuan; Chen, Xi; Sun, Wang; Li, Jiao

2018-01-01

Qinba area has a long history of tea planting and is a northernmost region in China where Camellia sinensis L. is grown. In order to provide basic data for selection and optimization of molecular markers of tea plants. 118 markers, including 40 EST-SSR, 40 SRAP and 38 SCoT markers were used to evaluate the genetic diversity of 50 tea plant ( Camellia sinensis. ) samples collected from Qinb. tea germplasm, assess population structure. In this study, a total of 414 alleles were obtained using 38 pairs of SCoT primers, with an average of 10.89 alleles per primer. The percentage of polymorphic bands (PPB), polymorphism information content (PIC), resolving power (Rp), effective multiplex ratio (EMR), average band informativeness (Ib av ), and marker index (MI) were 96.14%, 0.79, 6.71, 10.47, 0.58, and 6.07 respectively. 338 alleles were amplified via 40 pairs of SRAP (8.45 per primer), with PPB, PIC, Rp, EMR, Ib av, and MI values of 89.35%, 0.77, 5.11, 7.55, 0.61, and 4.61, respectively. Furthermore, 320 alleles have been detected using 40 EST-SSR primers (8.00 per primer), with PPB, PIC, Rp, EMR, Ib av , and MI values of 94.06%, 0.85, 4.48, 7.53, 0.56, and 4.22 respectively. These results indicated that SCoT markers had higher efficiency.Mantel test was used to analyze the genetic distance matrix generated by EST-SSRs, SRAPs and SCoTs. The results showed that the correlation between the genetic distance matrix based on EST-SSR and that based on SRAP was very small ( r  = 0.01), followed by SCoT and SRAP ( r  = 0.17), then by SCoT and EST-SSR ( r  = 0.19).The 50 tea samples were divided into two sub-populations using STRUCTURE, Neighbor-joining (NJ) method and principal component analyses (PCA). The results produced by STRUCTURE were completely consistent with the PCA analysis. Furthermore, there is no obvious relationship between the results produced using sub-populational and geographical data. Among the three types of markers, SCoT markers has many advantages in terms of NPB, PPB, Rp, EMR, and MI. Nevertheless, the values of PIC showed different trends, with the highest values generated with EST-SSR, followed by SCoT and SRAP. The average band informativeness showed similar trends. Correlation between genetic distances produced by three different molecular markers were very small, thus it is not recommended to use a single marker to evaluate genetic diversity and population structure. It is hence suggested that combining of different types of molecular markers should be used to evaluate the genetic diversity and population structure. It also seems crucial to screen out, for each type of molecular markers, core markers of Camellia sinensis . This study revealed that genes of exotic plant varieties have been constantly integrated into the gene pool of Qinba area tea. A low level of genetic diversity was observed; this is shown by an average coefficient of genetic similarity of 0.74.

The Chinese Air Force: Evolving Concepts, Roles, and Capabilities

DTIC Science & Technology

2012-01-01

5 The Missions of the People’s Liberation Army Air Force . . . 133 Murray Scot Tanner Chapter 6 The Development of the PLAAF’s Doctrine...the Military Region (MR) Headquarters. There are no indications this pattern of army domination will change in the next decade.” Murray Scot Tanner, a...his- tory, one can find remarkable threads of continuity. Early aircraft, though but fragile contraptions of wood and canvas, exploited the same

Chronic radiation exposure modifies temporal dynamics of cytogenetic but not reproductive indicators in Scots pine populations.

PubMed

Geras'kin, Stanislav; Oudalova, Alla; Kuzmenkov, Alexey; Vasiliyev, Denis

2018-04-18

Over a period of 13 years (2003-2015), reproductive and cytogenetic effects are investigated in Scots pine populations growing in the Bryansk region of Russia radioactively contaminated as a result of the Chernobyl accident. In reference populations, the frequencies of cytogenetic abnormalities are shown to change with time in a cyclic manner. In chronically exposed populations, the cyclic patterns in temporal dynamics of cytogenetic abnormalities appear to be disturbed. In addition, a tendency to decrease in the frequencies of cytogenetic abnormalities with time as well as an increase in their variability with dose rate is revealed. In contrast, no significant impact of chronic radiation exposure on the time dynamics of reproductive indexes is detected. Finally, long-term observations on chronically exposed Scots pine populations revealed qualitative differences in the temporal dynamics of reproductive and cytogenetic indicators. Copyright © 2018 Elsevier Ltd. All rights reserved.

Influence of long-term chronic exposure and weather conditions on Scots pine populations.

PubMed

Geras'kin, Stanislav; Vasiliyev, Denis; Makarenko, Ekaterina; Volkova, Polina; Kuzmenkov, Alexey

2017-04-01

Over a period of 8 years (2007-2014), we were evaluating seed quality and morphological abnormalities in Scots pine trees affected as a result of the Chernobyl accident. The calculated dose rates for the trees at the study sites varied from background values at the reference sites to 40 mGy/year at the most contaminated site. We investigated whether radioactive contamination and/or weather factors could decrease the reproductive capacity or increase the frequency of morphological abnormalities of needles in pine trees. Scots pine seeds are characterized by high interannual variability of viability, which is largely determined by weather conditions. No consistent differences in reproductive capacity were detected between the impacted and reference populations. Brachyblasts with three needles were found only in the affected populations; however, their frequency was very low and only at the very border of significance at the p < 0.10 level.

High resolution optical surface metrology with the slope measuring portable optical test system

NASA Astrophysics Data System (ADS)

Maldonado, Alejandro V.

New optical designs strive to achieve extreme performance, and continually increase the complexity of prescribed optical shapes, which often require wide dynamic range and high resolution. SCOTS, or the Software Configurable Optical Test System, can measure a wide range of optical surfaces with high sensitivity using surface slope. This dissertation introduces a high resolution version of SCOTS called SPOTS, or the Slope measuring Portable Optical Test System. SPOTS improves the metrology of surface features on the order of sub-millimeter to decimeter spatial scales and nanometer to micrometer level height scales. Currently there is no optical surface metrology instrument with the same utility. SCOTS uses a computer controlled display (such as an LCD monitor) and camera to measure surface slopes over the entire surface of a mirror. SPOTS differs in that an additional lens is placed near the surface under test. A small prototype system is discussed in general, providing the support for the design of future SPOTS devices. Then the SCOTS instrument transfer function is addressed, which defines the way the system filters surface heights. Lastly, the calibration and performance of larger SPOTS device is analyzed with example measurements of the 8.4-m diameter aspheric Large Synoptic Survey Telescope's (LSST) primary mirror. In general optical systems have a transfer function, which filters data. In the case of optical imaging systems the instrument transfer function (ITF) follows the modulation transfer function (MTF), which causes a reduction of contrast as a function of increasing spatial frequency due to diffraction. In SCOTS, ITF is shown to decrease the measured height of surface features as their spatial frequency increases, and thus the SCOTS and SPOTS ITF is proportional to their camera system's MTF. Theory and simulations are supported by a SCOTS measurement of a test piece with a set of lithographically written sinusoidal surface topographies. In addition, an example of a simple inverse filtering technique is provided. The success of a small SPOTS proof of concept instrument paved the way for a new larger prototype system, which is intended to measure subaperture regions on large optical mirrors. On large optics, the prototype SPOTS is light weight and it rests on the surface being tested. One advantage of this SPOTS is stability over time in maintaining its calibration. Thus the optician can simply place SPOTS on the mirror, perform a simple alignment, collect measurement data, then pick the system up and repeat at a new location. The entire process takes approximately 5 to 10 minutes, of which 3 minutes is spent collecting data. SPOTS' simplicity of design, light weight, robustness, wide dynamic range, and high sensitivity make it a useful tool for optical shop use during the fabrication and testing process of large and small optics.

Heavy metals accumulation in wood tissues of the forest-forming species growed in the Steppe technogenic landscapes in Ukraine

NASA Astrophysics Data System (ADS)

Lovinska, Viktoriia; Wiche, Oliver

2016-04-01

Territory of Steppe in Ukraine is affected by significant anthropogenic impact caused with mining, metallurgical, chemicalplants and heat power stations. The priority pollutants of the region emissions of these enterprises are presented such heavy metals as Cd, Pb, Cu, Zn, Ni, Mn. The regional forest ecosystems can be considered as potential concentrators of pollutants borned with different technogenic impact. It is necessary to study an ability of forests wood to accumulate heavy metals because accumulated toxins are eliminated from biogeochemical cycle in forest ecosystem for a long time. This study goal is to determine the accumulation properties of forest-forming species - Pinus sylvestris (Scots pine) and Robinia pseudoacacia (black locust) difference age group in relation to heavy metals. It was considerable also to assess the heavy metal distribution in the wood tissue of referred species.Heavy metals content were determined with atomic absorption spectrophotometer using. Scots pine and black locust are the main forest-forming species of natural and artificial forests within Northern Steppe.They can be seen as transformers of the heavy metals cycle and selective concentrators of toxic elements, under the conditions of their excessive concentrations in the environment.It was established that wood tissue of Scots pine and black locust accumulated cadmium in high concentrations according to the age in both species. Indexes of zinc accumulation in the wood of Scots pine exceeded the maximal value in the wood tissue of black locust. The results of our research demonstrated antagonistic interaction of cadmium and zinc. The highest copper concentrations was found for the trees at the age of 45 years. Lead has been identified in wood sample of all ages. Accumulation maximum was fixed in the oldest samples. The trend of concentration increasing of metal didn't find for both species. As for nickel there was established the opposite tendention for both studied species. Nickel maximum accumulation in black locust was fixed for the youngest copies. The same tendency was observed for Scots pine in copies of 45 years old. We did not see relation with age for mangan content in both studied species. The absolute value of the amount of mangan for Scots pine is much higher comparatevelly to black locust.

Optimization of routine KRAS mutation PCR-based testing procedure for rational individualized first-line-targeted therapy selection in metastatic colorectal cancer.

PubMed

Chretien, Anne-Sophie; Harlé, Alexandre; Meyer-Lefebvre, Magali; Rouyer, Marie; Husson, Marie; Ramacci, Carole; Harter, Valentin; Genin, Pascal; Leroux, Agnès; Merlin, Jean-Louis

2013-02-01

KRAS mutation detection represents a crucial issue in metastatic colorectal cancer (mCRC). The optimization of KRAS mutation detection delay enabling rational prescription of first-line treatment in mCRC including anti-EGFR-targeted therapy requires robust and rapid molecular biology techniques. Routine analysis of mutations in codons 12 and 13 on 674 paraffin-embedded tissue specimens of mCRC has been performed for KRAS mutations detection using three molecular biology techniques, that is, high-resolution melting (HRM), polymerase chain reaction restriction fragment length polymorphism (PCR-RFLP), and allelic discrimination PCR (TaqMan PCR). Discordant cases were assessed with COBAS 4800 KRAS CE-IVD assay. Among the 674 tumor specimens, 1.5% (10/674) had excessive DNA degradation and could not be analyzed. KRAS mutations were detected in 38.0% (256/674) of the analysable specimens (82.4% in codon 12 and 17.6% in codon 13). Among 613 specimens in whom all three techniques were used, 12 (2.0%) cases of discordance between the three techniques were observed. 83.3% (10/12) of the discordances were due to PCR-RFLP as confirmed by COBAS 4800 retrospective analysis. The three techniques were statistically comparable (κ > 0.9; P < 0.001). From these results, optimization of the routine procedure consisted of proceeding to systematic KRAS detection using HRM and TaqMan and PCR-RFLP in case of discordance and allowed significant decrease in delays. The results showed an excellent correlation between the three techniques. Using HRM and TaqMan warrants high-quality and rapid-routine KRAS mutation detection in paraffin-embedded tumor specimens. The new procedure allowed a significant decrease in delays for reporting results, enabling rational prescription of first-line-targeted therapy in mCRC.

Coast Guard Proceedings. Volume 70, Number 2, Summer 2013

DTIC Science & Technology

2013-01-01

the vil- lage of Point Hope, as the near-shore depths prevented Spar from anchoring close to the beach. Photo courtesy of Dr. Leslie Wood , U.S...Confer- ence, OTC Paper 909318, February 2011. Story, Jason, and Scot Tripp, lou Steinbrecher, Mike Sprague, Bob McKenna. Arctic Craft Investigation...Report. CG-D-02-12, RDC UDI #1215 Part A, August 2011. Story, Jason, and Scot Tripp, lou Steinbrecher, Mike Sprague, Bob McKenna. Arctic Craft

Twenty-two year results of a Scots pine (Pinus sylvestris L.) provenance test in North Dakota

Treesearch

Richard A. Cunningham; David F. Van Haverbeke

1991-01-01

A provenance test of 49 seed sources of Scots pine (Pinus sylvestris L.) from eastern Europe, Russia, and Siberia was established in two plantations in north-central North Dakota. After 22 years, trees from seed sources within the region bounded by 20° to 57° east longitude and 50° to 58° north latitude were taller, and larger in diameter, and had denser crown and...

Analysis, pretreatment and enzymatic saccharification of different fractions of Scots pine.

PubMed

Normark, Monica; Winestrand, Sandra; Lestander, Torbjörn A; Jönsson, Leif J

2014-03-19

Forestry residues consisting of softwood are a major lignocellulosic resource for production of liquid biofuels. Scots pine, a commercially important forest tree, was fractionated into seven fractions of chips: juvenile heartwood, mature heartwood, juvenile sapwood, mature sapwood, bark, top parts, and knotwood. The different fractions were characterized analytically with regard to chemical composition and susceptibility to dilute-acid pretreatment and enzymatic saccharification. All fractions were characterized by a high glucan content (38-43%) and a high content of other carbohydrates (11-14% mannan, 2-4% galactan) that generate easily convertible hexose sugars, and by a low content of inorganic material (0.2-0.9% ash). The lignin content was relatively uniform (27-32%) and the syringyl-guaiacyl ratio of the different fractions were within the range 0.021-0.025. The knotwood had a high content of extractives (9%) compared to the other fractions. The effects of pretreatment and enzymatic saccharification were relatively similar, but without pretreatment the bark fraction was considerably more susceptible to enzymatic saccharification. Since sawn timber is a main product from softwood species such as Scots pine, it is an important issue whether different parts of the tree are equally suitable for bioconversion processes. The investigation shows that bioconversion of Scots pine is facilitated by that most of the different fractions exhibit relatively similar properties with regard to chemical composition and susceptibility to techniques used for bioconversion of woody biomass.

Drought-induced defoliation and long periods of near-zero gas exchange play a key role in accentuating metabolic decline of Scots pine.

PubMed

Poyatos, Rafael; Aguadé, David; Galiano, Lucía; Mencuccini, Maurizio; Martínez-Vilalta, Jordi

2013-10-01

Drought-induced defoliation has recently been associated with the depletion of carbon reserves and increased mortality risk in Scots pine (Pinus sylvestris). We hypothesize that defoliated individuals are more sensitive to drought, implying that potentially higher gas exchange (per unit of leaf area) during wet periods may not compensate for their reduced photosynthetic area. We measured sap flow, needle water potentials and whole-tree hydraulic conductance to analyse the drought responses of co-occurring defoliated and nondefoliated Scots pines in northeast Spain during typical (2010) and extreme (2011) drought conditions. Defoliated Scots pines showed higher sap flow per unit leaf area during spring, but were more sensitive to summer drought, relative to nondefoliated pines. This pattern was associated with a steeper decline in soil-to-leaf hydraulic conductance with drought and an enhanced sensitivity of canopy conductance to soil water availability. Near-homeostasis in midday water potentials was observed across years and defoliation classes, with minimum values of -2.5 MPa. Enhanced sensitivity to drought and prolonged periods of near-zero gas exchange were consistent with low levels of carbohydrate reserves in defoliated trees. Our results support the critical links between defoliation, water and carbon availability, and their key roles in determining tree survival and recovery under drought. © 2013 The Authors. New Phytologist © 2013 New Phytologist Trust.

High-level expression of Camelid nanobodies in Nicotiana benthamiana.

PubMed

Teh, Yi-Hui Audrey; Kavanagh, Tony A

2010-08-01

Nanobodies (or VHHs) are single-domain antigen-binding fragments derived from Camelid heavy chain-only antibodies. Their small size, monomeric behaviour, high stability and solubility, and ability to bind epitopes not accessible to conventional antibodies make them especially suitable for many therapeutic and biotechnological applications. Here we describe high-level expression, in Nicotiana benthamiana, of three versions of an anti-hen egg white lysozyme (HEWL) nanobody which include the original VHH from an immunized library (cAbLys3), a codon-optimized derivative, and a codon-optimized hybrid nanobody comprising the CDRs of cAbLys3 grafted onto an alternative 'universal' nanobody framework. His6- and StrepII-tagged derivatives of each nanobody were targeted for accumulation in the cytoplasm, chloroplast and apoplast using different pre-sequences. When targeted to the apoplast, intact functional nanobodies accumulated at an exceptionally high level (up to 30% total leaf protein), demonstrating the great potential of plants as a nanobody production system.

Glyphosate resistance: state of knowledge

PubMed Central

Sammons, Robert Douglas; Gaines, Todd A

2014-01-01

Studies of mechanisms of resistance to glyphosate have increased current understanding of herbicide resistance mechanisms. Thus far, single-codon non-synonymous mutations of EPSPS (5-enolypyruvylshikimate-3-phosphate synthase) have been rare and, relative to other herbicide mode of action target-site mutations, unconventionally weak in magnitude for resistance to glyphosate. However, it is possible that weeds will emerge with non-synonymous mutations of two codons of EPSPS to produce an enzyme endowing greater resistance to glyphosate. Today, target-gene duplication is a common glyphosate resistance mechanism and could become a fundamental process for developing any resistance trait. Based on competition and substrate selectivity studies in several species, rapid vacuole sequestration of glyphosate occurs via a transporter mechanism. Conversely, as the chloroplast requires transporters for uptake of important metabolites, transporters associated with the two plastid membranes may separately, or together, successfully block glyphosate delivery. A model based on finite glyphosate dose and limiting time required for chloroplast loading sets the stage for understanding how uniquely different mechanisms can contribute to overall glyphosate resistance. PMID:25180399

Analysis of synonymous codon usage patterns in the genus Rhizobium.

PubMed

Wang, Xinxin; Wu, Liang; Zhou, Ping; Zhu, Shengfeng; An, Wei; Chen, Yu; Zhao, Lin

2013-11-01

The codon usage patterns of rhizobia have received increasing attention. However, little information is available regarding the conserved features of the codon usage patterns in a typical rhizobial genus. The codon usage patterns of six completely sequenced strains belonging to the genus Rhizobium were analysed as model rhizobia in the present study. The relative neutrality plot showed that selection pressure played a role in codon usage in the genus Rhizobium. Spearman's rank correlation analysis combined with correspondence analysis (COA) showed that the codon adaptation index and the effective number of codons (ENC) had strong correlation with the first axis of the COA, which indicated the important role of gene expression level and the ENC in the codon usage patterns in this genus. The relative synonymous codon usage of Cys codons had the strongest correlation with the second axis of the COA. Accordingly, the usage of Cys codons was another important factor that shaped the codon usage patterns in Rhizobium genomes and was a conserved feature of the genus. Moreover, the comparison of codon usage between highly and lowly expressed genes showed that 20 unique preferred codons were shared among Rhizobium genomes, revealing another conserved feature of the genus. This is the first report of the codon usage patterns in the genus Rhizobium.

A questionnaire to measure melanoma risk, knowledge and protective behaviour: assessing content validity in a convenience sample of Scots and Australians.

PubMed

Gillespie, Helen S; Watson, Tony; Emery, Jon D; Lee, Amanda J; Murchie, Peter

2011-08-25

The aim of this study was to assess the content validity of a questionnaire to measure melanoma risk, knowledge and protective behaviour in a convenience sample of Scots and Australians. Australia has the highest melanoma incidence worldwide but has developed a culture of skin cancer avoidance with a long history of skin cancer primary prevention campaigns of proven effectiveness. Scotland has lower incidence, but has shown a greater rate of increase between 1985 and 2007. There is an urgent need in Scotland, therefore, to identify those groups at greatest risk and provide them with effective preventative advice. A self-administered postal survey was completed by four groups formed from convenience samples in two geographical locations (Northeast Scotland and Western Australia). In univariate analysis scores on personal risk, level of concern, protective behaviour, and knowledge were compared by nationality, previous skin cancer diagnosis and personally knowing someone with melanoma. Multivariate linear regression analysis modelled the influence of potential predictor variables upon each of the scores. 540 people completed the questionnaire, 273 Scots (50.6%). 133 (24.6%) Scots and 83 (15.4%) Australians previously had melanoma or non-melanoma skin cancer, whilst 120 (22.2%) Scots and 190 (35.2%) Australians personally knew someone with melanoma. Australians had higher knowledge (p < 0.001), level of concern (p < 0.001) and protective behaviour (p < 0.001) scores than the Scottish. Australian nationality was the strongest independent predictor of a higher knowledge score (p < 0.001), followed by a previous skin cancer diagnosis (p = 0.003), personal knowledge of someone with melanoma (p = 0.011), female gender (p = 0.005) and higher education status (p < 0.001) (R(2) = 0.163). The questionnaire detected higher levels of knowledge and skin cancer protective behaviours in Australians than in Scottish people. This was expected and supports the content validity of the questionnaire and its value as a future research tool in the Scottish population.

The role of below-ground competition during early stages of secondary succession: the case of 3-year-old Scots pine (Pinus sylvestris L.) seedlings in an abandoned grassland.

PubMed

Picon-Cochard, Catherine; Coll, Lluis; Balandier, Philippe

2006-06-01

In abandoned or extensively managed grasslands, the mechanisms involved in pioneer tree species success are not fully explained. Resource competition among plants and microclimate modifications have been emphasised as possible mechanisms to explain variation of survivorship and growth. In this study, we evaluated a number of mechanisms that may lead to successful survival and growth of seedlings of a pioneer tree species (Pinus sylvestris) in a grass-dominated grassland. Three-year-old Scots pines were planted in an extensively managed grassland of the French Massif Central and for 2 years were either maintained in bare soil or subjected to aerial and below-ground interactions induced by grass vegetation. Soil temperatures were slightly higher in bare soil than under the grass vegetation, but not to an extent explaining pine growth differences. The tall grass canopy reduced light transmission by 77% at ground level and by 20% in the upper part of Scots pine seedlings. Grass vegetation presence also significantly decreased soil volumetric water content (Hv) and soil nitrate in spring and in summer. In these conditions, the average tree height was reduced by 5% compared to trees grown in bare soil, and plant biomass was reduced by 85%. Scots pine intrinsic water-use efficiency (A/g), measured by leaf gas-exchange, increased when Hv decreased owing to a rapid decline of stomatal conductance (g). This result was also confirmed by delta 13C analyses of needles. A summer 15N labelling of seedlings and grass vegetation confirmed the higher NO3 capture capacity of grass vegetation in comparison with Scots pine seedlings. Our results provide evidence that the seedlings' success was linked to tolerance of below-ground resource depletion (particularly water) induced by grass vegetation based on morphological and physiological plasticity as well as to resource conservation.

Genome-wide comparative analysis of codon usage bias and codon context patterns among cyanobacterial genomes.

PubMed

Prabha, Ratna; Singh, Dhananjaya P; Sinha, Swati; Ahmad, Khurshid; Rai, Anil

2017-04-01

With the increasing accumulation of genomic sequence information of prokaryotes, the study of codon usage bias has gained renewed attention. The purpose of this study was to examine codon selection pattern within and across cyanobacterial species belonging to diverse taxonomic orders and habitats. We performed detailed comparative analysis of cyanobacterial genomes with respect to codon bias. Our analysis reflects that in cyanobacterial genomes, A- and/or T-ending codons were used predominantly in the genes whereas G- and/or C-ending codons were largely avoided. Variation in the codon context usage of cyanobacterial genes corresponded to the clustering of cyanobacteria as per their GC content. Analysis of codon adaptation index (CAI) and synonymous codon usage order (SCUO) revealed that majority of genes are associated with low codon bias. Codon selection pattern in cyanobacterial genomes reflected compositional constraints as major influencing factor. It is also identified that although, mutational constraint may play some role in affecting codon usage bias in cyanobacteria, compositional constraint in terms of genomic GC composition coupled with environmental factors affected codon selection pattern in cyanobacterial genomes. Copyright © 2016 Elsevier B.V. All rights reserved.

Codon usage bias in prokaryotic pyrimidine-ending codons is associated with the degeneracy of the encoded amino acids

PubMed Central

Wald, Naama; Alroy, Maya; Botzman, Maya; Margalit, Hanah

2012-01-01

Synonymous codons are unevenly distributed among genes, a phenomenon termed codon usage bias. Understanding the patterns of codon bias and the forces shaping them is a major step towards elucidating the adaptive advantage codon choice can confer at the level of individual genes and organisms. Here, we perform a large-scale analysis to assess codon usage bias pattern of pyrimidine-ending codons in highly expressed genes in prokaryotes. We find a bias pattern linked to the degeneracy of the encoded amino acid. Specifically, we show that codon-pairs that encode two- and three-fold degenerate amino acids are biased towards the C-ending codon while codons encoding four-fold degenerate amino acids are biased towards the U-ending codon. This codon usage pattern is widespread in prokaryotes, and its strength is correlated with translational selection both within and between organisms. We show that this bias is associated with an improved correspondence with the tRNA pool, avoidance of mis-incorporation errors during translation and moderate stability of codon–anticodon interaction, all consistent with more efficient translation. PMID:22581775

The helicase Ded1p controls use of near-cognate translation initiation codons in 5' UTRs.

PubMed

Guenther, Ulf-Peter; Weinberg, David E; Zubradt, Meghan M; Tedeschi, Frank A; Stawicki, Brittany N; Zagore, Leah L; Brar, Gloria A; Licatalosi, Donny D; Bartel, David P; Weissman, Jonathan S; Jankowsky, Eckhard

2018-06-27

The conserved and essential DEAD-box RNA helicase Ded1p from yeast and its mammalian orthologue DDX3 are critical for the initiation of translation 1 . Mutations in DDX3 are linked to tumorigenesis 2-4 and intellectual disability 5 , and the enzyme is targeted by a range of viruses 6 . How Ded1p and its orthologues engage RNAs during the initiation of translation is unknown. Here we show, by integrating transcriptome-wide analyses of translation, RNA structure and Ded1p-RNA binding, that the effects of Ded1p on the initiation of translation are connected to near-cognate initiation codons in 5' untranslated regions. Ded1p associates with the translation pre-initiation complex at the mRNA entry channel and repressing the activity of Ded1p leads to the accumulation of RNA structure in 5' untranslated regions, the initiation of translation from near-cognate start codons immediately upstream of these structures and decreased protein synthesis from the corresponding main open reading frames. The data reveal a program for the regulation of translation that links Ded1p, the activation of near-cognate start codons and mRNA structure. This program has a role in meiosis, in which a marked decrease in the levels of Ded1p is accompanied by the activation of the alternative translation initiation sites that are seen when the activity of Ded1p is repressed. Our observations indicate that Ded1p affects translation initiation by controlling the use of near-cognate initiation codons that are proximal to mRNA structure in 5' untranslated regions.

Optimizing doped libraries by using genetic algorithms

NASA Astrophysics Data System (ADS)

Tomandl, Dirk; Schober, Andreas; Schwienhorst, Andreas

1997-01-01

The insertion of random sequences into protein-encoding genes in combination with biologicalselection techniques has become a valuable tool in the design of molecules that have usefuland possibly novel properties. By employing highly effective screening protocols, a functionaland unique structure that had not been anticipated can be distinguished among a hugecollection of inactive molecules that together represent all possible amino acid combinations.This technique is severely limited by its restriction to a library of manageable size. Oneapproach for limiting the size of a mutant library relies on `doping schemes', where subsetsof amino acids are generated that reveal only certain combinations of amino acids in a proteinsequence. Three mononucleotide mixtures for each codon concerned must be designed, suchthat the resulting codons that are assembled during chemical gene synthesis represent thedesired amino acid mixture on the level of the translated protein. In this paper we present adoping algorithm that `reverse translates' a desired mixture of certain amino acids into threemixtures of mononucleotides. The algorithm is designed to optimally bias these mixturestowards the codons of choice. This approach combines a genetic algorithm with localoptimization strategies based on the downhill simplex method. Disparate relativerepresentations of all amino acids (and stop codons) within a target set can be generated.Optional weighing factors are employed to emphasize the frequencies of certain amino acidsand their codon usage, and to compensate for reaction rates of different mononucleotidebuilding blocks (synthons) during chemical DNA synthesis. The effect of statistical errors thataccompany an experimental realization of calculated nucleotide mixtures on the generatedmixtures of amino acids is simulated. These simulations show that the robustness of differentoptima with respect to small deviations from calculated values depends on their concomitantfitness. Furthermore, the calculations probe the fitness landscape locally and allow apreliminary assessment of its structure.

Ribosomal scanning past the primary initiation codon as a mechanism for expression of CTL epitopes encoded in alternative reading frames

PubMed Central

1996-01-01

An increasing amount of evidence has shown that epitopes restricted to MHC class I molecules and recognized by CTL need not be encoded in a primary open reading frame (ORF). Such epitopes have been demonstrated after stop codons, in alternative reading frames (RF) and within introns. We have used a series of frameshifts (FS) introduced into the Influenza A/PR/8 /34 nucleoprotein (NP) gene to confirm the previous in vitro observations of cryptic epitope expression, and show that they are sufficiently expressed to prime immune responses in vivo. This presentation is not due to sub-dominant epitopes, transcription from cryptic promoters beyond the point of the FS, or internal initiation of translation. By introducing additional mutations to the construct exhibiting the most potent presentation, we have identified initiation codon readthrough (termed scanthrough here, where the scanning ribosome bypasses the conventional initiation codon, initiating translation further downstream) as the likely mechanism of epitope production. Further mutational analysis demonstrated that, while it should operate during the expression of wild-type (WT) protein, scanthrough does not provide a major source of processing substrate in our system. These findings suggest (i) that the full array of self- and pathogen-derived epitopes available during thymic selection and infection has not been fully appreciated and (ii) that cryptic epitope expression should be considered when the specificity of a CTL response cannot be identified or in therapeutic situations when conventional CTL targets are limited, as may be the case with latent viral infections and transformed cells. Finally, initiation codon readthrough provides a plausible explanation for the presentation of exocytic proteins by MHC class I molecules. PMID:8879204

[Protein S3 in the human 80S ribosome adjoins mRNA from 3'-side of the A-site codon].

PubMed

Molotkov, M V; Graĭfer, D M; Popugaeva, E A; Bulygin, K N; Meshchaninova, M I; Ven'iaminova, A G; Karpova, G G

2007-01-01

The protein environment of mRNA 3' of the A-site codon (the decoding site) in the human 80S ribosome was studied using a set of oligoribonucleotide derivatives bearing a UUU triplet at the 5'-end and a perfluoroarylazide group at one of the nucleotide residues at the 3'-end of this triplet. Analogues of mRNA were phased into the ribosome using binding at the tRNAPhe P-site, which recognizes the UUU codon. Mild UV irradiation of ribosome complexes with tRNAPhe and mRNA analogues resulted in the predominant crosslinking of the analogues with the 40S subunit components, mainly with proteins and, to a lesser extent, with rRNA. Among the 40S subunit ribosomal proteins, the S3 protein was the main target for modification in all cases. In addition, minor crosslinking with the S2 protein was observed. The crosslinking with the S3 and S2 proteins occurred both in triple complexes and in the absence of tRNA. Within triple complexes, crosslinking with S15 protein was also found, its efficiency considerably falling when the modified nucleotide was moved from positions +5 to +12 relative to the first codon nucleotide in the P-site. In some cases, crosslinking with the S30 protein was observed, it was most efficient for the derivative containing a photoreactive group at the +7 adenosine residue. The results indicate that the S3 protein in the human ribosome plays a key role in the formation of the mRNA binding site 3' of the codon in the decoding site.

Discursive Maps at the Edge of Chaos

DTIC Science & Technology

2017-05-25

study. As Arthur Herman explains in How the Scots Invented the Modern World, much of this was the product of the Scottish revolution.20 The French...Routledge, 2004), 77. 20 Arthur Herman, How The Scots Invented The Modern World (New York: Crown Publishers, 2001), 62-85. 21 Thomas S. Kuhn, The...kochruli.htm#fn41. 26 Denis Wood and John Fels, The Power Of Maps (New York: Guilford Press, 1992), 46. 11 The Semantic Turn, “meaning does not exist all

The Roots of the Command and Control of Air Power: An Appraisal of Three Air Forces Through 1945

DTIC Science & Technology

2014-09-17

Co., 1927), 121. 33 Joubert de la Ferté, The Third Service, 48. 34 Scot Robertson, The Development of RAF Strategic Bombing Doctrine, 1919-1939...5 Scot Robertson, The Development of RAF Strategic Bombing Doctrine, 1919-1939 (Westport, Conn: Praeger, 1995), 36. 6 Powers, Strategy without...Royal United Services Institution Vol LXXV (February 1930): 109. 36 Collier, The Defence of the United Kingdom, 12.; Derek Wood and Derek D. Dempster

Integrated analysis of individual codon contribution to protein biosynthesis reveals a new approach to improving the basis of rational gene design

PubMed Central

Villada, Juan C.; Brustolini, Otávio José Bernardes

2017-01-01

Abstract Gene codon optimization may be impaired by the misinterpretation of frequency and optimality of codons. Although recent studies have revealed the effects of codon usage bias (CUB) on protein biosynthesis, an integrated perspective of the biological role of individual codons remains unknown. Unlike other previous studies, we show, through an integrated framework that attributes of codons such as frequency, optimality and positional dependency should be combined to unveil individual codon contribution for protein biosynthesis. We designed a codon quantification method for assessing CUB as a function of position within genes with a novel constraint: the relativity of position-dependent codon usage shaped by coding sequence length. Thus, we propose a new way of identifying the enrichment, depletion and non-uniform positional distribution of codons in different regions of yeast genes. We clustered codons that shared attributes of frequency and optimality. The cluster of non-optimal codons with rare occurrence displayed two remarkable characteristics: higher codon decoding time than frequent–non-optimal cluster and enrichment at the 5′-end region, where optimal codons with the highest frequency are depleted. Interestingly, frequent codons with non-optimal adaptation to tRNAs are uniformly distributed in the Saccharomyces cerevisiae genes, suggesting their determinant role as a speed regulator in protein elongation. PMID:28449100

Integrated analysis of individual codon contribution to protein biosynthesis reveals a new approach to improving the basis of rational gene design.

PubMed

Villada, Juan C; Brustolini, Otávio José Bernardes; Batista da Silveira, Wendel

2017-08-01

Gene codon optimization may be impaired by the misinterpretation of frequency and optimality of codons. Although recent studies have revealed the effects of codon usage bias (CUB) on protein biosynthesis, an integrated perspective of the biological role of individual codons remains unknown. Unlike other previous studies, we show, through an integrated framework that attributes of codons such as frequency, optimality and positional dependency should be combined to unveil individual codon contribution for protein biosynthesis. We designed a codon quantification method for assessing CUB as a function of position within genes with a novel constraint: the relativity of position-dependent codon usage shaped by coding sequence length. Thus, we propose a new way of identifying the enrichment, depletion and non-uniform positional distribution of codons in different regions of yeast genes. We clustered codons that shared attributes of frequency and optimality. The cluster of non-optimal codons with rare occurrence displayed two remarkable characteristics: higher codon decoding time than frequent-non-optimal cluster and enrichment at the 5'-end region, where optimal codons with the highest frequency are depleted. Interestingly, frequent codons with non-optimal adaptation to tRNAs are uniformly distributed in the Saccharomyces cerevisiae genes, suggesting their determinant role as a speed regulator in protein elongation. © The Author 2017. Published by Oxford University Press on behalf of Kazusa DNA Research Institute.

Partial attenuation of Marek's disease virus by manipulation of Di-codon bias

USDA-ARS?s Scientific Manuscript database

All species studied to date demonstrate a preference for certain codons over other synonymous codons (codon bias), a preference which is also observed for pairs of codons (di-codon bias). Previous studies using poliovirus and influenza virus as models have demonstrated the ability to cause attenuat...

Transcriptome Analysis of Core Dinoflagellates Reveals a Universal Bias towards "GC" Rich Codons.

PubMed

Williams, Ernest; Place, Allen; Bachvaroff, Tsvetan

2017-04-27

Although dinoflagellates are a potential source of pharmaceuticals and natural products, the mechanisms for regulating and producing these compounds are largely unknown because of extensive post-transcriptional control of gene expression. One well-documented mechanism for controlling gene expression during translation is codon bias, whereby specific codons slow or even terminate protein synthesis. Approximately 10,000 annotatable genes from fifteen "core" dinoflagellate transcriptomes along a range of overall guanine and cytosine (GC) content were used for codonW analysis to determine the relative synonymous codon usage (RSCU) and the GC content at each codon position. GC bias in the analyzed dataset and at the third codon position varied from 51% and 54% to 66% and 88%, respectively. Codons poor in GC were observed to be universally absent, but bias was most pronounced for codons ending in uracil followed by adenine (UA). GC bias at the third codon position was able to explain low abundance codons as well as the low effective number of codons. Thus, we propose that a bias towards codons rich in GC bases is a universal feature of core dinoflagellates, possibly relating to their unique chromosome structure, and not likely a major mechanism for controlling gene expression.

Fluid self-diffusion in Scots pine sapwood tracheid cells.

PubMed

Johannessen, Espen H; Hansen, Eddy W; Rosenholm, Jarl B

2006-02-09

The self-diffusion coefficients of water and toluene in Scots pine sapwood was measured using low field pulsed field gradient nuclear magnetic resonance (PFG-NMR). Wood chips of 8 mm diameter were saturated with the respective liquids, and liquid self-diffusion was then traced in one dimension orthogonal to the tracheid cell walls in the wood's radial direction. The experimental echo attenuation curves were exponential, and characteristic self-diffusion coefficients were produced for diffusion times spanning from very short times to times on the order of magnitude of seconds. Observed self-diffusion coefficients were decaying asymptotically as a function of diffusion time, an effect which was ascribed to the cell walls' restriction on confined liquid diffusion. The observed self-diffusion behavior in Scots pine sapwood was compared to self-diffusion coefficients obtained from simulations of diffusion in a square. Principles of molecular displacements in confined geometries were used for elucidating the wood's cellular structure from the observed diffusion coefficients. The results were compared with a mathematical model for diffusion between parallel planes.

Multiple Evolutionary Selections Involved in Synonymous Codon Usages in the Streptococcus agalactiae Genome.

PubMed

Ma, Yan-Ping; Ke, Hao; Liang, Zhi-Ling; Liu, Zhen-Xing; Hao, Le; Ma, Jiang-Yao; Li, Yu-Gu

2016-02-24

Streptococcus agalactiae is an important human and animal pathogen. To better understand the genetic features and evolution of S. agalactiae, multiple factors influencing synonymous codon usage patterns in S. agalactiae were analyzed in this study. A- and U-ending rich codons were used in S. agalactiae function genes through the overall codon usage analysis, indicating that Adenine (A)/Thymine (T) compositional constraints might contribute an important role to the synonymous codon usage pattern. The GC3% against the effective number of codon (ENC) value suggested that translational selection was the important factor for codon bias in the microorganism. Principal component analysis (PCA) showed that (i) mutational pressure was the most important factor in shaping codon usage of all open reading frames (ORFs) in the S. agalactiae genome; (ii) strand specific mutational bias was not capable of influencing the codon usage bias in the leading and lagging strands; and (iii) gene length was not the important factor in synonymous codon usage pattern in this organism. Additionally, the high correlation between tRNA adaptation index (tAI) value and codon adaptation index (CAI), frequency of optimal codons (Fop) value, reinforced the role of natural selection for efficient translation in S. agalactiae. Comparison of synonymous codon usage pattern between S. agalactiae and susceptible hosts (human and tilapia) showed that synonymous codon usage of S. agalactiae was independent of the synonymous codon usage of susceptible hosts. The study of codon usage in S. agalactiae may provide evidence about the molecular evolution of the bacterium and a greater understanding of evolutionary relationships between S. agalactiae and its hosts.

Multiple Evolutionary Selections Involved in Synonymous Codon Usages in the Streptococcus agalactiae Genome

PubMed Central

Ma, Yan-Ping; Ke, Hao; Liang, Zhi-Ling; Liu, Zhen-Xing; Hao, Le; Ma, Jiang-Yao; Li, Yu-Gu

2016-01-01

Streptococcus agalactiae is an important human and animal pathogen. To better understand the genetic features and evolution of S. agalactiae, multiple factors influencing synonymous codon usage patterns in S. agalactiae were analyzed in this study. A- and U-ending rich codons were used in S. agalactiae function genes through the overall codon usage analysis, indicating that Adenine (A)/Thymine (T) compositional constraints might contribute an important role to the synonymous codon usage pattern. The GC3% against the effective number of codon (ENC) value suggested that translational selection was the important factor for codon bias in the microorganism. Principal component analysis (PCA) showed that (i) mutational pressure was the most important factor in shaping codon usage of all open reading frames (ORFs) in the S. agalactiae genome; (ii) strand specific mutational bias was not capable of influencing the codon usage bias in the leading and lagging strands; and (iii) gene length was not the important factor in synonymous codon usage pattern in this organism. Additionally, the high correlation between tRNA adaptation index (tAI) value and codon adaptation index (CAI), frequency of optimal codons (Fop) value, reinforced the role of natural selection for efficient translation in S. agalactiae. Comparison of synonymous codon usage pattern between S. agalactiae and susceptible hosts (human and tilapia) showed that synonymous codon usage of S. agalactiae was independent of the synonymous codon usage of susceptible hosts. The study of codon usage in S. agalactiae may provide evidence about the molecular evolution of the bacterium and a greater understanding of evolutionary relationships between S. agalactiae and its hosts. PMID:26927064

Translation efficiencies of synonymous codons are not always correlated with codon usage in tobacco chloroplasts.

PubMed

Nakamura, Masayuki; Sugiura, Masahiro

2007-01-01

Codon usage in chloroplasts is different from that in prokaryotic and eukaryotic nuclear genomes. However, no experimental approach has been made to analyse the translation efficiency of individual codons in chloroplasts. We devised an in vitro assay for translation efficiencies using synthetic mRNAs, and measured the translation efficiencies of five synonymous codon groups in tobacco chloroplasts. Among four alanine codons (GCN, where N is U, C, A or G), GCU was the most efficient for translation, whereas the chloroplast genome lacks tRNA genes corresponding to GCU. Phenylalanine and tyrosine are each encoded by two codons (UUU/C and UAU/C, respectively). Phenylalanine UUC and tyrosine UAC were translated more than twice as efficiently than UUU and UAU, respectively, contrary to their codon usage, whereas translation efficiencies of synonymous codons for alanine, aspartic acid and asparagine were parallel to their codon usage. These observations indicate that translation efficiencies of individual codons are not always correlated with codon usage in vitro in chloroplasts. This raises an important issue for foreign gene expression in chloroplasts.

Analysis, pretreatment and enzymatic saccharification of different fractions of Scots pine

PubMed Central

2014-01-01

Background Forestry residues consisting of softwood are a major lignocellulosic resource for production of liquid biofuels. Scots pine, a commercially important forest tree, was fractionated into seven fractions of chips: juvenile heartwood, mature heartwood, juvenile sapwood, mature sapwood, bark, top parts, and knotwood. The different fractions were characterized analytically with regard to chemical composition and susceptibility to dilute-acid pretreatment and enzymatic saccharification. Results All fractions were characterized by a high glucan content (38-43%) and a high content of other carbohydrates (11-14% mannan, 2-4% galactan) that generate easily convertible hexose sugars, and by a low content of inorganic material (0.2-0.9% ash). The lignin content was relatively uniform (27-32%) and the syringyl-guaiacyl ratio of the different fractions were within the range 0.021-0.025. The knotwood had a high content of extractives (9%) compared to the other fractions. The effects of pretreatment and enzymatic saccharification were relatively similar, but without pretreatment the bark fraction was considerably more susceptible to enzymatic saccharification. Conclusions Since sawn timber is a main product from softwood species such as Scots pine, it is an important issue whether different parts of the tree are equally suitable for bioconversion processes. The investigation shows that bioconversion of Scots pine is facilitated by that most of the different fractions exhibit relatively similar properties with regard to chemical composition and susceptibility to techniques used for bioconversion of woody biomass. PMID:24641769

Chloroplast DNA codon use: evidence for selection at the psb A locus based on tRNA availability.

PubMed

Morton, B R

1993-09-01

Codon use in the three sequenced chloroplast genomes (Marchantia, Oryza, and Nicotiana) is examined. The chloroplast has a bias in that codons NNA and NNT are favored over synonymous NNC and NNG codons. This appears to be a consequence of an overall high A + T content of the genome. This pattern of codon use is not followed by the psb A gene of all three genomes and other psb A sequences examined. In this gene, the codon use favors NNC over NNT for twofold degenerate amino acids. In each case the only tRNA coded by the genome is complementary to the NNC codon. This codon use is similar to the codon use by chloroplast genes examined from Chlamydomonas reinhardtii. Since psb A is the major translation product of the chloroplast, this suggests that selection is acting on the codon use of this gene to adapt codons to tRNA availability, as previously suggested for unicellular organisms.

Genomic analysis of codon usage shows influence of mutation pressure, natural selection, and host features on Marburg virus evolution.

PubMed

Nasrullah, Izza; Butt, Azeem M; Tahir, Shifa; Idrees, Muhammad; Tong, Yigang

2015-08-26

The Marburg virus (MARV) has a negative-sense single-stranded RNA genome, belongs to the family Filoviridae, and is responsible for several outbreaks of highly fatal hemorrhagic fever. Codon usage patterns of viruses reflect a series of evolutionary changes that enable viruses to shape their survival rates and fitness toward the external environment and, most importantly, their hosts. To understand the evolution of MARV at the codon level, we report a comprehensive analysis of synonymous codon usage patterns in MARV genomes. Multiple codon analysis approaches and statistical methods were performed to determine overall codon usage patterns, biases in codon usage, and influence of various factors, including mutation pressure, natural selection, and its two hosts, Homo sapiens and Rousettus aegyptiacus. Nucleotide composition and relative synonymous codon usage (RSCU) analysis revealed that MARV shows mutation bias and prefers U- and A-ended codons to code amino acids. Effective number of codons analysis indicated that overall codon usage among MARV genomes is slightly biased. The Parity Rule 2 plot analysis showed that GC and AU nucleotides were not used proportionally which accounts for the presence of natural selection. Codon usage patterns of MARV were also found to be influenced by its hosts. This indicates that MARV have evolved codon usage patterns that are specific to both of its hosts. Moreover, selection pressure from R. aegyptiacus on the MARV RSCU patterns was found to be dominant compared with that from H. sapiens. Overall, mutation pressure was found to be the most important and dominant force that shapes codon usage patterns in MARV. To our knowledge, this is the first detailed codon usage analysis of MARV and extends our understanding of the mechanisms that contribute to codon usage and evolution of MARV.

Transcriptome Analysis of Core Dinoflagellates Reveals a Universal Bias towards “GC” Rich Codons

PubMed Central

Williams, Ernest; Place, Allen; Bachvaroff, Tsvetan

2017-01-01

Although dinoflagellates are a potential source of pharmaceuticals and natural products, the mechanisms for regulating and producing these compounds are largely unknown because of extensive post-transcriptional control of gene expression. One well-documented mechanism for controlling gene expression during translation is codon bias, whereby specific codons slow or even terminate protein synthesis. Approximately 10,000 annotatable genes from fifteen “core” dinoflagellate transcriptomes along a range of overall guanine and cytosine (GC) content were used for codonW analysis to determine the relative synonymous codon usage (RSCU) and the GC content at each codon position. GC bias in the analyzed dataset and at the third codon position varied from 51% and 54% to 66% and 88%, respectively. Codons poor in GC were observed to be universally absent, but bias was most pronounced for codons ending in uracil followed by adenine (UA). GC bias at the third codon position was able to explain low abundance codons as well as the low effective number of codons. Thus, we propose that a bias towards codons rich in GC bases is a universal feature of core dinoflagellates, possibly relating to their unique chromosome structure, and not likely a major mechanism for controlling gene expression. PMID:28448468

Characterization of the porcine epidemic diarrhea virus codon usage bias.

PubMed

Chen, Ye; Shi, Yuzhen; Deng, Hongjuan; Gu, Ting; Xu, Jian; Ou, Jinxin; Jiang, Zhiguo; Jiao, Yiren; Zou, Tan; Wang, Chong

2014-12-01

Porcine epidemic diarrhea virus (PEDV) has been responsible for several recent outbreaks of porcine epidemic diarrhea (PED) and has caused great economic loss in the swine-raising industry. Considering the significance of PEDV, a systemic analysis was performed to study its codon usage patterns. The relative synonymous codon usage value of each codon revealed that codon usage bias exists and that PEDV tends to use codons that end in T. The mean ENC value of 47.91 indicates that the codon usage bias is low. However, we still wanted to identify the cause of this codon usage bias. A correlation analysis between the codon compositions (A3s, T3s, G3s, C3s, and GC3s), the ENC values, and the nucleotide contents (A%, T%, G%, C%, and GC%) indicated that mutational bias plays role in shaping the PEDV codon usage bias. This was further confirmed by a principal component analysis between the codon compositions and the axis values. Using the Gravy, Aroma, and CAI values, a role of natural selection in the PEDV codon usage pattern was also identified. Neutral analysis indicated that natural selection pressure plays a more important role than mutational bias in codon usage bias. Natural selection also plays an increasingly significant role during PEDV evolution. Additionally, gene function and geographic distribution also influence the codon usage bias to a degree. Copyright © 2014 Elsevier B.V. All rights reserved.

Developing a Zebrafish Model of NF1 for Structure-Function Analysis and Identification of Modifier Genes

DTIC Science & Technology

2012-04-01

nuclease (ZFN) technology (Figure 34). We were fortunate to have the opportunity to collaborate closely with the laboratories Nathan Lawson and Scot Wolfe...controls tumour cell growth. Nature 441, 424–430. 32. Postlethwait, J.H., Woods , I.G., Ngo-Hazelett, P., Yan, Y.L., Kelly, P.D., Chu, F., Huang, H...Michael Granato2, Nathan D. Lawson4, Scot A. Wolfe4, Seok-Hyung Kim5, Lilianna Solnica- Krezel6, John P. Kanki1, Keith L. Ligon3, Jonathan A. Epstein2

Chronic radiation exposure as an ecological factor: Hypermethylation and genetic differentiation in irradiated Scots pine populations.

PubMed

Volkova, P Yu; Geras'kin, S A; Horemans, N; Makarenko, E S; Saenen, E; Duarte, G T; Nauts, R; Bondarenko, V S; Jacobs, G; Voorspoels, S; Kudin, M

2018-01-01

Genetic and epigenetic changes were investigated in chronically irradiated Scots pine (Pinus sylvestris L.) populations from territories that were heavily contaminated by radionuclides as result of the Chernobyl Nuclear Power Plant accident. In comparison to the reference site, the genetic diversity revealed by electrophoretic mobility of AFLPs was found to be significantly higher at the radioactively contaminated areas. In addition, the genome of pine trees was significantly hypermethylated at 4 of the 7 affected sites. Copyright © 2017 Elsevier Ltd. All rights reserved.

Genome-wide analysis of codon usage bias in Ebolavirus.

PubMed

Cristina, Juan; Moreno, Pilar; Moratorio, Gonzalo; Musto, Héctor

2015-01-22

Ebola virus (EBOV) is a member of the family Filoviridae and its genome consists of a 19-kb, single-stranded, negative sense RNA. EBOV is subdivided into five distinct species with different pathogenicities, being Zaire ebolavirus (ZEBOV) the most lethal species. The interplay of codon usage among viruses and their hosts is expected to affect overall viral survival, fitness, evasion from host's immune system and evolution. In the present study, we performed comprehensive analyses of codon usage and composition of ZEBOV. Effective number of codons (ENC) indicates that the overall codon usage among ZEBOV strains is slightly biased. Different codon preferences in ZEBOV genes in relation to codon usage of human genes were found. Highly preferred codons are all A-ending triplets, which strongly suggests that mutational bias is a main force shaping codon usage in ZEBOV. Dinucleotide composition also plays a role in the overall pattern of ZEBOV codon usage. ZEBOV does not seem to use the most abundant tRNAs present in the human cells for most of their preferred codons. Copyright © 2014 Elsevier B.V. All rights reserved.

Crenolanib is a type I tyrosine kinase inhibitor that inhibits mutant KIT D816 isoforms prevalent in systemic mastocytosis and core binding factor leukemia.

PubMed

Kampa-Schittenhelm, Kerstin Maria; Frey, Julia; Haeusser, Lara A; Illing, Barbara; Pavlovsky, Ashly A; Blumenstock, Gunnar; Schittenhelm, Marcus Matthias

2017-10-10

Activating D816 mutations of the class III receptor tyrosine kinase KIT are associated with the majority of patients with systemic mastocytosis (SM), but also core binding factor (CBF) AML, making KIT mutations attractive therapeutic targets for the treatment of these cancers. Crenolanib is a potent and selective inhibitor of wild-type as well as mutant isoforms of the class III receptor tyrosine kinases FLT3 and PDGFRα/β. Notably, crenolanib inhibits constitutively active mutant-FLT3 isoforms resulting from amino acid substitutions of aspartic acid at codon 835, which is homologous to codon 816 in the KIT gene - suggesting sensitivity against mutant-KIT D816 isoforms as well. Here we demonstrate that crenolanib targets KIT D816 in SM and CBF AML models: crenolanib inhibits cellular proliferation and initiates apoptosis of mastocytosis cell lines expressing these mutations. Target-specificity was confirmed using an isogenic cell model. In addition, we demonstrate that KIT D816 mutations are targetable with clinically achievable doses of crenolanib. Further, a rationale to combine cladribine (2-CDA), the therapeutic standard in SM, with crenolanib is provided. In conclusion, we demonstrate that crenolanib is an inhibitor of mutant-KIT D816 isoforms at clinically achievable concentrations, and thus may be a potential treatment for SM and CBF AML as a monotherapy or in combination approaches.

Molecular mimicry of human tRNALys anti-codon domain by HIV-1 RNA genome facilitates tRNA primer annealing.

PubMed

Jones, Christopher P; Saadatmand, Jenan; Kleiman, Lawrence; Musier-Forsyth, Karin

2013-02-01

The primer for initiating reverse transcription in human immunodeficiency virus type 1 (HIV-1) is tRNA(Lys3). Host cell tRNA(Lys) is selectively packaged into HIV-1 through a specific interaction between the major tRNA(Lys)-binding protein, human lysyl-tRNA synthetase (hLysRS), and the viral proteins Gag and GagPol. Annealing of the tRNA primer onto the complementary primer-binding site (PBS) in viral RNA is mediated by the nucleocapsid domain of Gag. The mechanism by which tRNA(Lys3) is targeted to the PBS and released from hLysRS prior to annealing is unknown. Here, we show that hLysRS specifically binds to a tRNA anti-codon-like element (TLE) in the HIV-1 genome, which mimics the anti-codon loop of tRNA(Lys) and is located proximal to the PBS. Mutation of the U-rich sequence within the TLE attenuates binding of hLysRS in vitro and reduces the amount of annealed tRNA(Lys3) in virions. Thus, LysRS binds specifically to the TLE, which is part of a larger LysRS binding domain in the viral RNA that includes elements of the Psi packaging signal. Our results suggest that HIV-1 uses molecular mimicry of the anti-codon of tRNA(Lys) to increase the efficiency of tRNA(Lys3) annealing to viral RNA.

Molecular mimicry of human tRNALys anti-codon domain by HIV-1 RNA genome facilitates tRNA primer annealing

PubMed Central

Jones, Christopher P.; Saadatmand, Jenan; Kleiman, Lawrence; Musier-Forsyth, Karin

2013-01-01

The primer for initiating reverse transcription in human immunodeficiency virus type 1 (HIV-1) is tRNALys3. Host cell tRNALys is selectively packaged into HIV-1 through a specific interaction between the major tRNALys-binding protein, human lysyl-tRNA synthetase (hLysRS), and the viral proteins Gag and GagPol. Annealing of the tRNA primer onto the complementary primer-binding site (PBS) in viral RNA is mediated by the nucleocapsid domain of Gag. The mechanism by which tRNALys3 is targeted to the PBS and released from hLysRS prior to annealing is unknown. Here, we show that hLysRS specifically binds to a tRNA anti-codon-like element (TLE) in the HIV-1 genome, which mimics the anti-codon loop of tRNALys and is located proximal to the PBS. Mutation of the U-rich sequence within the TLE attenuates binding of hLysRS in vitro and reduces the amount of annealed tRNALys3 in virions. Thus, LysRS binds specifically to the TLE, which is part of a larger LysRS binding domain in the viral RNA that includes elements of the Psi packaging signal. Our results suggest that HIV-1 uses molecular mimicry of the anti-codon of tRNALys to increase the efficiency of tRNALys3 annealing to viral RNA. PMID:23264568

Minigene-like inhibition of protein synthesis mediated by hungry codons near the start codon

PubMed Central

Jacinto-Loeza, Eva; Vivanco-Domínguez, Serafín; Guarneros, Gabriel; Hernández-Sánchez, Javier

2008-01-01

Rare AGA or AGG codons close to the initiation codon inhibit protein synthesis by a tRNA-sequestering mechanism as toxic minigenes do. To further understand this mechanism, a parallel analysis of protein synthesis and peptidyl-tRNA accumulation was performed using both a set of lacZ constructs where AGAAGA codons were moved codon by codon from +2, +3 up to +7, +8 positions and a series of 3–8 codon minigenes containing AGAAGA codons before the stop codon. β-Galactosidase synthesis from the AGAAGA lacZ constructs (in a Pth defective in vitro system without exogenous tRNA) diminished as the AGAAGA codons were closer to AUG codon. Likewise, β-galactosidase expression from the reporter +7 AGA lacZ gene (plus tRNA, 0.25 μg/μl) waned as the AGAAGAUAA minigene shortened. Pth counteracted both the length-dependent minigene effect on the expression of β-galactosidase from the +7 AGA lacZ reporter gene and the positional effect from the AGAAGA lacZ constructs. The +2, +3 AGAAGA lacZ construct and the shortest +2, +3 AGAAGAUAA minigene accumulated the highest percentage of peptidyl-tRNAArg4. These observations lead us to propose that hungry codons at early positions, albeit with less strength, inhibit protein synthesis by a minigene-like mechanism involving accumulation of peptidyl-tRNA. PMID:18583364

Synonymous codon usage of genes in polymerase complex of Newcastle disease virus.

PubMed

Kumar, Chandra Shekhar; Kumar, Sachin

2017-06-01

Newcastle disease virus (NDV) is pathogenic to both avian and non-avian species but extensively finds poultry as its primary host and causes heavy economic losses in the poultry industry. In this study, a total of 186 polymerase complex comprising of nucleoprotein (N), phosphoprotein (P), and large polymerase (L) genes of NDV was analyzed for synonymous codon usage. The relative synonymous codon usage and effective number of codons (ENC) values were used to estimate codon usage variation in each gene. Correspondence analysis (COA) was used to study the major trend in codon usage variation. Analyzing the ENC plot values against GC3s (at synonymous third codon position) we concluded that mutational pressure was the main factor determining codon usage bias than translational selection in NDV N, P, and L genes. Moreover, correlation analysis indicated, that aromaticity of N, P, and L genes also influenced the codon usage variation. The varied distribution of pathotypes for N, P, and L gene clearly suggests that change in codon usage for NDV is pathotype specific. The codon usage preference similarity in N, P, and L gene might be detrimental for polymerase complex functioning. The study represents a comprehensive analysis to date of N, P, and L genes codon usage pattern of NDV and provides a basic understanding of the mechanisms for codon usage bias. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Evolution of Synonymous Codon Usage in Neurospora tetrasperma and Neurospora discreta

PubMed Central

Whittle, C. A.; Sun, Y.; Johannesson, H.

2011-01-01

Neurospora comprises a primary model system for the study of fungal genetics and biology. In spite of this, little is known about genome evolution in Neurospora. For example, the evolution of synonymous codon usage is largely unknown in this genus. In the present investigation, we conducted a comprehensive analysis of synonymous codon usage and its relationship to gene expression and gene length (GL) in Neurospora tetrasperma and Neurospora discreta. For our analysis, we examined codon usage among 2,079 genes per organism and assessed gene expression using large-scale expressed sequenced tag (EST) data sets (279,323 and 453,559 ESTs for N. tetrasperma and N. discreta, respectively). Data on relative synonymous codon usage revealed 24 codons (and two putative codons) that are more frequently used in genes with high than with low expression and thus were defined as optimal codons. Although codon-usage bias was highly correlated with gene expression, it was independent of selectively neutral base composition (introns); thus demonstrating that translational selection drives synonymous codon usage in these genomes. We also report that GL (coding sequences [CDS]) was inversely associated with optimal codon usage at each gene expression level, with highly expressed short genes having the greatest frequency of optimal codons. Optimal codon frequency was moderately higher in N. tetrasperma than in N. discreta, which might be due to variation in selective pressures and/or mating systems. PMID:21402862

Codon usage bias in phylum Actinobacteria: relevance to environmental adaptation and host pathogenicity.

PubMed

Lal, Devi; Verma, Mansi; Behura, Susanta K; Lal, Rup

2016-10-01

Actinobacteria are Gram-positive bacteria commonly found in soil, freshwater and marine ecosystems. In this investigation, bias in codon usages of ninety actinobacterial genomes was analyzed by estimating different indices of codon bias such as Nc (effective number of codons), SCUO (synonymous codon usage order), RSCU (relative synonymous codon usage), as well as sequence patterns of codon contexts. The results revealed several characteristic features of codon usage in Actinobacteria, as follows: 1) C- or G-ending codons are used frequently in comparison with A- and U ending codons; 2) there is a direct relationship of GC content with use of specific amino acids such as alanine, proline and glycine; 3) there is an inverse relationship between GC content and Nc estimates, 4) there is low SCUO value (<0.5) for most genes; and 5) GCC-GCC, GCC-GGC, GCC-GAG and CUC-GAC are the frequent context sequences among codons. This study highlights the fact that: 1) in Actinobacteria, extreme GC content and codon bias are driven by mutation rather than natural selection; (2) traits like aerobicity are associated with effective natural selection and therefore low GC content and low codon bias, demonstrating the role of both mutational bias and translational selection in shaping the habitat and phenotype of actinobacterial species. Copyright © 2016 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.

Analysis of transcriptome data reveals multifactor constraint on codon usage in Taenia multiceps.

PubMed

Huang, Xing; Xu, Jing; Chen, Lin; Wang, Yu; Gu, Xiaobin; Peng, Xuerong; Yang, Guangyou

2017-04-20

Codon usage bias (CUB) is an important evolutionary feature in genomes that has been widely observed in many organisms. However, the synonymous codon usage pattern in the genome of T. multiceps remains to be clarified. In this study, we analyzed the codon usage of T. multiceps based on the transcriptome data to reveal the constraint factors and to gain an improved understanding of the mechanisms that shape synonymous CUB. Analysis of a total of 8,620 annotated mRNA sequences from T. multiceps indicated only a weak codon bias, with mean GC and GC3 content values of 49.29% and 51.43%, respectively. Our analysis indicated that nucleotide composition, mutational pressure, natural selection, gene expression level, amino acids with grand average of hydropathicity (GRAVY) and aromaticity (Aromo) and the effective selection of amino-acids all contributed to the codon usage in T. multiceps. Among these factors, natural selection was implicated as the major factor affecting the codon usage variation in T. multiceps. The codon usage of ribosome genes was affected mainly by mutations, while the essential genes were affected mainly by selection. In addition, 21codons were identified as "optimal codons". Overall, the optimal codons were GC-rich (GC:AU, 41:22), and ended with G or C (except CGU). Furthermore, different degrees of variation in codon usage were found between T. multiceps and Escherichia coli, yeast, Homo sapiens. However, little difference was found between T. multiceps and Taenia pisiformis. In this study, the codon usage pattern of T. multiceps was analyzed systematically and factors affected CUB were also identified. This is the first study of codon biology in T. multiceps. Understanding the codon usage pattern in T. multiceps can be helpful for the discovery of new genes, molecular genetic engineering and evolutionary studies.

Codon usage patterns in Nematoda: analysis based on over 25 million codons in thirty-two species

PubMed Central

2006-01-01

Background Codon usage has direct utility in molecular characterization of species and is also a marker for molecular evolution. To understand codon usage within the diverse phylum Nematoda, we analyzed a total of 265,494 expressed sequence tags (ESTs) from 30 nematode species. The full genomes of Caenorhabditis elegans and C. briggsae were also examined. A total of 25,871,325 codons were analyzed and a comprehensive codon usage table for all species was generated. This is the first codon usage table available for 24 of these organisms. Results Codon usage similarity in Nematoda usually persists over the breadth of a genus but then rapidly diminishes even within each clade. Globodera, Meloidogyne, Pristionchus, and Strongyloides have the most highly derived patterns of codon usage. The major factor affecting differences in codon usage between species is the coding sequence GC content, which varies in nematodes from 32% to 51%. Coding GC content (measured as GC3) also explains much of the observed variation in the effective number of codons (R = 0.70), which is a measure of codon bias, and it even accounts for differences in amino acid frequency. Codon usage is also affected by neighboring nucleotides (N1 context). Coding GC content correlates strongly with estimated noncoding genomic GC content (R = 0.92). On examining abundant clusters in five species, candidate optimal codons were identified that may be preferred in highly expressed transcripts. Conclusion Evolutionary models indicate that total genomic GC content, probably the product of directional mutation pressure, drives codon usage rather than the converse, a conclusion that is supported by examination of nematode genomes. PMID:26271136

A detailed analysis of codon usage patterns and influencing factors in Zika virus.

PubMed

Singh, Niraj K; Tyagi, Anuj

2017-07-01

Recent outbreaks of Zika virus (ZIKV) in Africa, Latin America, Europe, and Southeast Asia have resulted in serious health concerns. To understand more about evolution and transmission of ZIKV, detailed codon usage analysis was performed for all available strains. A high effective number of codons (ENC) value indicated the presence of low codon usage bias in ZIKV. The effect of mutational pressure on codon usage bias was confirmed by significant correlations between nucleotide compositions at third codon positions and ENCs. Correlation analysis between Gravy values, Aroma values and nucleotide compositions at third codon positions also indicated some influence of natural selection. However, the low codon adaptation index (CAI) value of ZIKV with reference to human and mosquito indicated poor adaptation of ZIKV codon usage towards its hosts, signifying that natural selection has a weaker influence than mutational pressure. Additionally, relative dinucleotide frequencies, geographical distribution, and evolutionary processes also influenced the codon usage pattern to some extent.

Abolition of Peroxiredoxin-5 Mitochondrial Targeting during Canid Evolution

PubMed Central

Van der Eecken, Valérie; Clippe, André; Dekoninck, Sophie; Goemaere, Julie; Walbrecq, Geoffroy; Van Veldhoven, Paul P.; Knoops, Bernard

2013-01-01

In human, the subcellular targeting of peroxiredoxin-5 (PRDX5), a thioredoxin peroxidase, is dependent on the use of multiple alternative transcription start sites and two alternative in-frame translation initiation sites, which determine whether or not the region encoding a mitochondrial targeting sequence (MTS) is translated. In the present study, the abolition of PRDX5 mitochondrial targeting in dog is highlighted and the molecular mechanism underlying the loss of mitochondrial PRDX5 during evolution is examined. Here, we show that the absence of mitochondrial PRDX5 is generalized among the extant canids and that the first events leading to PRDX5 MTS abolition in canids involve a mutation in the more 5′ translation initiation codon as well as the appearance of a STOP codon. Furthermore, we found that PRDX5 MTS functionality is maintained in giant panda and northern elephant seal, which are phylogenetically closely related to canids. Also, the functional consequences of the restoration of mitochondrial PRDX5 in dog Madin-Darby canine kidney (MDCK) cells were investigated. The restoration of PRDX5 mitochondrial targeting in MDCK cells, instead of protecting, provokes deleterious effects following peroxide exposure independently of its peroxidase activity, indicating that mitochondrial PRDX5 gains cytotoxic properties under acute oxidative stress in MDCK cells. Altogether our results show that, although mitochondrial PRDX5 cytoprotective function against oxidative stress has been clearly demonstrated in human and rodents, PRDX5 targeting to mitochondria has been evolutionary lost in canids. Moreover, restoration of mitochondrial PRDX5 in dog MDCK cells, instead of conferring protection against peroxide exposure, makes them more vulnerable. PMID:24023783

Switches in Genomic GC Content Drive Shifts of Optimal Codons under Sustained Selection on Synonymous Sites

PubMed Central

Sun, Yu; Tamarit, Daniel

2017-01-01

Abstract The major codon preference model suggests that codons read by tRNAs in high concentrations are preferentially utilized in highly expressed genes. However, the identity of the optimal codons differs between species although the forces driving such changes are poorly understood. We suggest that these questions can be tackled by placing codon usage studies in a phylogenetic framework and that bacterial genomes with extreme nucleotide composition biases provide informative model systems. Switches in the background substitution biases from GC to AT have occurred in Gardnerella vaginalis (GC = 32%), and from AT to GC in Lactobacillus delbrueckii (GC = 62%) and Lactobacillus fermentum (GC = 63%). We show that despite the large effects on codon usage patterns by these switches, all three species evolve under selection on synonymous sites. In G. vaginalis, the dramatic codon frequency changes coincide with shifts of optimal codons. In contrast, the optimal codons have not shifted in the two Lactobacillus genomes despite an increased fraction of GC-ending codons. We suggest that all three species are in different phases of an on-going shift of optimal codons, and attribute the difference to a stronger background substitution bias and/or longer time since the switch in G. vaginalis. We show that comparative and correlative methods for optimal codon identification yield conflicting results for genomes in flux and discuss possible reasons for the mispredictions. We conclude that switches in the direction of the background substitution biases can drive major shifts in codon preference patterns even under sustained selection on synonymous codon sites. PMID:27540085

Dynamic Modeling of GAIT System Reveals Transcriptome Expansion and Translational Trickle Control Device

PubMed Central

Yao, Peng; Potdar, Alka A.; Arif, Abul; Ray, Partho Sarothi; Mukhopadhyay, Rupak; Willard, Belinda; Xu, Yichi; Yan, Jun; Saidel, Gerald M.; Fox, Paul L.

2012-01-01

SUMMARY Post-transcriptional regulatory mechanisms superimpose “fine-tuning” control upon “on-off” switches characteristic of gene transcription. We have exploited computational modeling with experimental validation to resolve an anomalous relationship between mRNA expression and protein synthesis. Differential GAIT (Gamma-interferon Activated Inhibitor of Translation) complex activation repressed VEGF-A synthesis to a low, constant rate despite high, variable VEGFA mRNA expression. Dynamic model simulations indicated the presence of an unidentified, inhibitory GAIT element-interacting factor. We discovered a truncated form of glutamyl-prolyl tRNA synthetase (EPRS), the GAIT constituent that binds the 3’-UTR GAIT element in target transcripts. The truncated protein, EPRSN1, prevents binding of functional GAIT complex. EPRSN1 mRNA is generated by a remarkable polyadenylation-directed conversion of a Tyr codon in the EPRS coding sequence to a stop codon (PAY*). By low-level protection of GAIT element-bearing transcripts, EPRSN1 imposes a robust “translational trickle” of target protein expression. Genome-wide analysis shows PAY* generates multiple truncated transcripts thereby contributing to transcriptome expansion. PMID:22386318

Use of signal sequences as an in situ removable sequence element to stimulate protein synthesis in cell-free extracts

PubMed Central

Ahn, Jin-Ho; Hwang, Mi-Yeon; Lee, Kyung-Ho; Choi, Cha-Yong; Kim, Dong-Myung

2007-01-01

This study developed a method to boost the expression of recombinant proteins in a cell-free protein synthesis system without leaving additional amino acid residues. It was found that the nucleotide sequences of the signal peptides serve as an efficient downstream box to stimulate protein synthesis when they were fused upstream of the target genes. The extent of stimulation was critically affected by the identity of the second codons of the signal sequences. Moreover, the yield of the synthesized protein was enhanced by as much as 10 times in the presence of an optimal second codon. The signal peptides were in situ cleaved and the target proteins were produced in their native sizes by carrying out the cell-free synthesis reactions in the presence of Triton X-100, most likely through the activation of signal peptidase in the S30 extract. The amplification of the template DNA and the addition of the signal sequences were accomplished by PCR. Hence, elevated levels of recombinant proteins were generated within several hours. PMID:17185295

Canopy Defoliation has More Impact on Carbohydrate Availability than on Hydraulic Function in Declining Scots Pine Populations

NASA Astrophysics Data System (ADS)

Poyatos, R.; Aguadé, D.; Gómez, M.; Mencuccini, M.; Martínez-Vilalta, J.

2013-12-01

Drought-induced defoliation has recently been associated with depletion of carbohydrate reserves and increased mortality risk in Scots pine (Pinus sylvestris L.) at its dry limit. Are defoliated pines hydraulically impaired compared to non-defoliated pines? Moreover, how do defoliated pines cope with potentially lethal droughts, as compared to non-defoliated pines in the same population? In order to address these questions, we measured the seasonal dynamics of sap flow and needle water potentials (2010-2012), hydraulic function and non-structural carbohydrates (NSC) (2012) in healthy and defoliated pines in the Prades mountains (NE Spain). The summer drought was mild in 2010, intense in 2012 and extremely long in 2011. Defoliated Scots pines showed higher sap flow per unit leaf area during spring, but they were more sensitive to summer drought (Figure 1). This pattern was associated with a steeper decline in soil-to-leaf hydraulic conductance, which could not be explained by differences in branch vulnerability to embolism across defoliation classes. Accordingly, the native loss of xylem conductivity in branches, measured in 2012, remained similar across defoliation classes and reached >65% at the peak of the drought. However, a steeper vulnerability curve was observed for root xylem of defoliated pines. Xylem diameter variations (2011-2012) will be used to further investigate possible differences in the aboveground/belowground partitioning of hydraulic resistance across defoliation classes. NSC levels varied across tree organs (leaves>branches>roots>trunk) and strongly declined with drought. Defoliated pines displayed reduced NSC levels throughout the study period, despite enhanced water transport capacity and increased gas exchange rates during spring. Overall, the defoliated vs. healthy status seems to be more associated to differences in carbohydrate storage and dynamics than to hydraulic differences per se. However, starch conversion to soluble sugars during drought also suggests that NSC may be actively involved in the maintenance of xylem and phloem transport. These results highlight the close connection between carbon and water relations in declining Scots pines. Seasonal course (2010-2012) of VPD (upper panel), soil moisture (mid panel) and sap flow per unit leaf area of defoliated and non defoliated Scots pines (lower panel).

Impacts of changing climate on the productivity of Norway spruce dominant stands with a mixture of Scots pine and birch in relation to water availability in southern and northern Finland.

PubMed

Ge, Zhen-Ming; Kellomäki, Seppo; Peltola, Heli; Zhou, Xiao; Wang, Kai-Yun; Väisänen, Hannu

2011-03-01

A process-based ecosystem model was used to assess the impacts of changing climate on net photosynthesis and total stem wood growth in relation to water availability in two unmanaged Norway spruce (Picea abies) dominant stands with a mixture of Scots pine (Pinus sylvestris) and birch (Betula sp.). The mixed stands were grown over a 100-year rotation (2000-99) in southern and northern Finland with initial species shares of 50, 25 and 25% for Norway spruce, Scots pine and birch, respectively. In addition, pure Norway spruce, Scots pine and birch stands were used as a comparison to identify whether species' response is different in mixed and pure stands. Soil type and moisture conditions (moderate drought) were expected to be the same at the beginning of the simulations irrespective of site location. Regardless of tree species, both annual net canopy photosynthesis (P(nc)) and total stem wood growth (V(s)) were, on average, lower on the southern site under the changing climate compared with the current climate (difference increasing toward the end of the rotation); the opposite was the case for the northern site. Regarding the stand water budget, evapotranspiration (E(T)) was higher under the changing climate regardless of site location. Transpiration and evaporation from the canopy affected water depletion the most. Norway spruce and birch accounted for most of the water depletion in mixed stands on both sites regardless of climatic condition. The annual soil water deficit (W(d)) was higher on the southern site under the changing climate. On the northern site, the situation was the opposite. According to our results, the growth of pure Norway spruce stands in southern Finland could be even lower than the growth of Norway spruce in mixed stands under the changing climate. The opposite was found for pure Scots pine and birch stands due to lower water depletion. This indicates that in the future the management should be properly adapted to climate change in order to sustain the productivity of mixed stands dominated by Norway spruce.

Structural and climatic determinants of demographic rates of Scots pine forests across the Iberian Peninsula.

PubMed

Vilà-Cabrera, Albert; Martínez-Vilalta, Jordi; Vayreda, Jordi; Retana, Javier

2011-06-01

The demographic rates of tree species typically show large spatial variation across their range. Understanding the environmental factors underlying this variation is a key topic in forest ecology, with far-reaching management implications. Scots pine (Pinus sylvestris L.) covers large areas of the Northern Hemisphere, the Iberian Peninsula being its southwestern distribution limit. In recent decades, an increase in severe droughts and a densification of forests as a result of changes in forest uses have occurred in this region. Our aim was to use climate and stand structure data to explain mortality and growth patterns of Scots pine forests across the Iberian Peninsula. We used data from 2392 plots dominated by Scots pine, sampled for the National Forest Inventory of Spain. Plots were sampled from 1986 to 1996 (IFN2) and were resampled from 1997 to 2007 (IFN3), allowing for the calculation of growth and mortality rates. We fitted linear models to assess the response of growth and mortality rates to the spatial variability of climate, climatic anomalies, and forest structure. Over the period of approximately 10 years between the IFN2 and IFN3, the amount of standing dead trees increased 11-fold. Higher mortality rates were related to dryness, and growth was reduced with increasing dryness and temperature, but results also suggested that effects of climatic stressors were not restricted to dry sites only. Forest structure was strongly related to demographic rates, suggesting that stand development and competition are the main factors associated with demography. In the case of mortality, forest structure interacted with climate, suggesting that competition for water resources induces tree mortality in dry sites. A slight negative relationship was found between mortality and growth, indicating that both rates are likely to be affected by the same stress factors. Additionally, regeneration tended to be lower in plots with higher mortality. Taken together, our results suggest a large-scale self-thinning related to the recent densification of Scots pine forests. This process appears to be enhanced by dry conditions and may lead to a mismatch in forest turnover. Forest management may be an essential adaptive tool under the drier conditions predicted by most climate models.

Assessing forest vulnerability to climate warming using a process-based model of tree growth: bad prospects for rear-edges.

PubMed

Sánchez-Salguero, Raúl; Camarero, Jesus Julio; Gutiérrez, Emilia; González Rouco, Fidel; Gazol, Antonio; Sangüesa-Barreda, Gabriel; Andreu-Hayles, Laia; Linares, Juan Carlos; Seftigen, Kristina

2017-07-01

Growth models can be used to assess forest vulnerability to climate warming. If global warming amplifies water deficit in drought-prone areas, tree populations located at the driest and southernmost distribution limits (rear-edges) should be particularly threatened. Here, we address these statements by analyzing and projecting growth responses to climate of three major tree species (silver fir, Abies alba; Scots pine, Pinus sylvestris; and mountain pine, Pinus uncinata) in mountainous areas of NE Spain. This region is subjected to Mediterranean continental conditions, it encompasses wide climatic, topographic and environmental gradients, and, more importantly, it includes rear-edges of the continuous distributions of these tree species. We used tree-ring width data from a network of 110 forests in combination with the process-based Vaganov-Shashkin-Lite growth model and climate-growth analyses to forecast changes in tree growth during the 21st century. Climatic projections were based on four ensembles CO 2 emission scenarios. Warm and dry conditions during the growing season constrain silver fir and Scots pine growth, particularly at the species rear-edge. By contrast, growth of high-elevation mountain pine forests is enhanced by climate warming. The emission scenario (RCP 8.5) corresponding to the most pronounced warming (+1.4 to 4.8 °C) forecasted mean growth reductions of -10.7% and -16.4% in silver fir and Scots pine, respectively, after 2050. This indicates that rising temperatures could amplify drought stress and thus constrain the growth of silver fir and Scots pine rear-edge populations growing at xeric sites. Contrastingly, mountain pine growth is expected to increase by +12.5% due to a longer and warmer growing season. The projections of growth reduction in silver fir and Scots pine portend dieback and a contraction of their species distribution areas through potential local extinctions of the most vulnerable driest rear-edge stands. Our modeling approach provides accessible tools to evaluate forest vulnerability to warmer conditions. © 2016 John Wiley & Sons Ltd.

Shore platform downwearing in eastern Canada; A 9-14 year micro-erosion meter record

NASA Astrophysics Data System (ADS)

Trenhaile, Alan S.; Porter, Neil J.

2018-06-01

Downwearing rates (erosion in the vertical plane) were measured with a micro-erosion meter (MEM) in eastern Canada, on an argillacious, sub-horizontal shore platform at Mont Louis in eastern Québec, and on two sloping, basaltic and sandstone platforms at, respectively, Scots Bay and Burntcoat Head in the Bay of Fundy, Nova Scotia. The original data covered a period from 2002 to 2009. This dataset was extended by measurements repeated at surviving MEM stations in 2017, producing records ranging over 9-14 years, depending on when each station was installed. Because of rapid surface downwearing, many of the original MEM stations were inoperable in 2017, especially at Burntcoat Head. Nevertheless, data were obtained from 19 stations at Burntcoat (35% of the 2009 original), 25 at Mont Louis (83% of the original), and 38 at Scots Bay (75% of the original). For the stations at Mont Louis and Scots Bay that were still functioning in 2017, there were no significant differences in rates of downwearing over the shorter (from station installation up to 2009) and extended periods (from installation to 2017). Mean rates of downwearing calculated from all the stations in each area declined through time, however, due to the loss of the more rapidly eroding stations. A simple procedure, which was proposed to compensate for this decrease, produced mean downwearing rates that were broadly similar to those reported over the original measurement period. There were significant relationships between downwearing rates and elevation (R2 = 0.32) and downwearing rates and rock hardness (R2 = 0.41) in the extended record at Scots Bay, and a small but significant relationship between downwearing rates and rock hardness at Mont Louis (R2 = 0.17). Differences in downwearing rates across the platforms suggest that salt weathering and wetting and drying are dominant weathering mechanisms at Scots Bay and Mont Louis. Chemical weathering of the sandstone cementing agent and the premature removal of weathered grains by wave-generated bottom currents may, however, be more important at Burntcoat Head.

Fine root biomass in relation to site and stand characteristics in Norway spruce and Scots pine stands.

PubMed

Helmisaari, Heljä-Sisko; Derome, John; Nöjd, Pekka; Kukkola, Mikko

2007-10-01

Variations in fine root biomass of trees and understory in 16 stands throughout Finland were examined and relationships to site and stand characteristics determined. Norway spruce fine root biomass varied between 184 and 370 g m(-2), and that of Scots pine ranged between 149 and 386 g m(-2). In northern Finland, understory roots and rhizomes (< 2 mm diameter) accounted for up to 50% of the stand total fine root biomass. Therefore, the fine root biomass of trees plus understory was larger in northern Finland in stands of both tree species, resulting in a negative relationship between fine root biomass and the temperature sum and a positive relationship between fine root biomass and the carbon:nitrogen ratio of the soil organic layer. The foliage:fine root ratio varied between 2.1 and 6.4 for Norway spruce and between 0.8 and 2.2 for Scots pine. The ratio decreased for both Norway spruce and Scots pine from south to north, as well as from fertile to more infertile site types. The foliage:fine root ratio of Norway spruce was related to basal area and stem surface area. The strong positive correlations of these three parameters with fine root nitrogen concentration implies that more fine roots are needed to maintain a certain amount of foliage when nutrient availability is low. No significant relationships were found between stand parameters and fine root biomass at the stand level, but the relationships considerably improved when both fine root biomass and stand parameters were calculated for the mean tree in the stand. When the northern and southern sites were analyzed separately, fine root biomass per tree of both species was significantly correlated with basal area and stem surface area per tree. Basal area, stem surface area and stand density can be estimated accurately and easily. Thus, our results may have value in predicting fine root biomass at the tree and stand level in boreal Norway spruce and Scots pine forests.

CodonLogo: a sequence logo-based viewer for codon patterns.

PubMed

Sharma, Virag; Murphy, David P; Provan, Gregory; Baranov, Pavel V

2012-07-15

Conserved patterns across a multiple sequence alignment can be visualized by generating sequence logos. Sequence logos show each column in the alignment as stacks of symbol(s) where the height of a stack is proportional to its informational content, whereas the height of each symbol within the stack is proportional to its frequency in the column. Sequence logos use symbols of either nucleotide or amino acid alphabets. However, certain regulatory signals in messenger RNA (mRNA) act as combinations of codons. Yet no tool is available for visualization of conserved codon patterns. We present the first application which allows visualization of conserved regions in a multiple sequence alignment in the context of codons. CodonLogo is based on WebLogo3 and uses the same heuristics but treats codons as inseparable units of a 64-letter alphabet. CodonLogo can discriminate patterns of codon conservation from patterns of nucleotide conservation that appear indistinguishable in standard sequence logos. The CodonLogo source code and its implementation (in a local version of the Galaxy Browser) are available at http://recode.ucc.ie/CodonLogo and through the Galaxy Tool Shed at http://toolshed.g2.bx.psu.edu/.

Forced Ambiguity of the Leucine Codons for Multiple-Site-Specific Incorporation of a Noncanonical Amino Acid

PubMed Central

Kwon, Inchan; Choi, Eun Sil

2016-01-01

Multiple-site-specific incorporation of a noncanonical amino acid into a recombinant protein would be a very useful technique to generate multiple chemical handles for bioconjugation and multivalent binding sites for the enhanced interaction. Previously combination of a mutant yeast phenylalanyl-tRNA synthetase variant and the yeast phenylalanyl-tRNA containing the AAA anticodon was used to incorporate a noncanonical amino acid into multiple UUU phenylalanine (Phe) codons in a site-specific manner. However, due to the less selective codon recognition of the AAA anticodon, there was significant misincorporation of a noncanonical amino acid into unwanted UUC Phe codons. To enhance codon selectivity, we explored degenerate leucine (Leu) codons instead of Phe degenerate codons. Combined use of the mutant yeast phenylalanyl-tRNA containing the CAA anticodon and the yPheRS_naph variant allowed incorporation of a phenylalanine analog, 2-naphthylalanine, into murine dihydrofolate reductase in response to multiple UUG Leu codons, but not to other Leu codon sites. Despite the moderate UUG codon occupancy by 2-naphthylalaine, these results successfully demonstrated that the concept of forced ambiguity of the genetic code can be achieved for the Leu codons, available for multiple-site-specific incorporation. PMID:27028506

Forced Ambiguity of the Leucine Codons for Multiple-Site-Specific Incorporation of a Noncanonical Amino Acid.

PubMed

Kwon, Inchan; Choi, Eun Sil

2016-01-01

Multiple-site-specific incorporation of a noncanonical amino acid into a recombinant protein would be a very useful technique to generate multiple chemical handles for bioconjugation and multivalent binding sites for the enhanced interaction. Previously combination of a mutant yeast phenylalanyl-tRNA synthetase variant and the yeast phenylalanyl-tRNA containing the AAA anticodon was used to incorporate a noncanonical amino acid into multiple UUU phenylalanine (Phe) codons in a site-specific manner. However, due to the less selective codon recognition of the AAA anticodon, there was significant misincorporation of a noncanonical amino acid into unwanted UUC Phe codons. To enhance codon selectivity, we explored degenerate leucine (Leu) codons instead of Phe degenerate codons. Combined use of the mutant yeast phenylalanyl-tRNA containing the CAA anticodon and the yPheRS_naph variant allowed incorporation of a phenylalanine analog, 2-naphthylalanine, into murine dihydrofolate reductase in response to multiple UUG Leu codons, but not to other Leu codon sites. Despite the moderate UUG codon occupancy by 2-naphthylalaine, these results successfully demonstrated that the concept of forced ambiguity of the genetic code can be achieved for the Leu codons, available for multiple-site-specific incorporation.

Impact of summer drought on isoprenoid emissions and carbon sink of three Scots pine provenances

PubMed Central

Lüpke, M.; Leuchner, M.; Steinbrecher, R.; Menzel, A.

2016-01-01

Scots pine (Pinus sylvestris L.) provenances cover broad ecological amplitudes. In a greenhouse study, we investigated the impact of drought stress and rewetting on gas exchange for three provenances (Italy: Emilia Romagna; Spain: Alto Ebro; Germany: East-German lowlands) of 2-year old Scots pine seedlings. CO2, water vapour and isoprenoid exchange of stressed and control trees were quantified with a four-chamber dynamic-enclosure system in the controlled environment of a climate chamber. The three provenances showed distinct isoprenoid emission patterns and were classified into a non-Δ3-carene, with either high α-/β-pinene or β-myrcene fraction, and a Δ3-carene dominated type. Isoprenoid emission rates, net-photosynthesis and transpiration were reduced during summer drought stress and significantly recovered after rewetting. A seasonal increase of isoprenoid emission rates towards autumn was observed for all control groups. Compared with the German provenance, the Spanish and Italian provenances revealed higher isoprenoid emission rates and more plastic responses to drought stress and seasonal development, which points to a local adaptation to climate. As a result of drought, net carbon uptake and transpiration of trees was reduced, but recovered after rewetting. We conclude from our study that Scots pine isoprenoid emission is more variable than expected and sensitive to drought periods, likely impacting regional air chemistry. Thus, a provenance-specific emission assessment accounting for reduced emission during prolonged (summer) drought is recommend for setting up biogenic volatile organic compound emission inventories used in air quality models. PMID:27591438

Intraspecific variability in functional traits matters: case study of Scots pine.

PubMed

Laforest-Lapointe, Isabelle; Martínez-Vilalta, Jordi; Retana, Javier

2014-08-01

Although intraspecific trait variability is an important component of species ecological plasticity and niche breadth, its implications for community and functional ecology have not been thoroughly explored. We characterized the intraspecific functional trait variability of Scots pine (Pinus sylvestris) in Catalonia (NE Spain) in order to (1) compare it to the interspecific trait variability of trees in the same region, (2) explore the relationships among functional traits and the relationships between them and stand and climatic variables, and (3) study the role of functional trait variability as a determinant of radial growth. We considered five traits: wood density (WD), maximum tree height (H max), leaf nitrogen content (Nmass), specific leaf area (SLA), and leaf biomass-to-sapwood area ratio (B L:A S). A unique dataset was obtained from the Ecological and Forest Inventory of Catalonia (IEFC), including data from 406 plots. Intraspecific trait variation was substantial for all traits, with coefficients of variation ranging between 8% for WD and 24% for B L:A S. In some cases, correlations among functional traits differed from those reported across species (e.g., H max and WD were positively related, whereas SLA and Nmass were uncorrelated). Overall, our model accounted for 47% of the spatial variability in Scots pine radial growth. Our study emphasizes the hierarchy of factors that determine intraspecific variations in functional traits in Scots pine and their strong association with spatial variability in radial growth. We claim that intraspecific trait variation is an important determinant of responses of plants to changes in climate and other environmental factors, and should be included in predictive models of vegetation dynamics.

Impact of summer drought on isoprenoid emissions and carbon sink of three Scots pine provenances.

PubMed

Lüpke, M; Leuchner, M; Steinbrecher, R; Menzel, A

2016-11-01

Scots pine (Pinus sylvestris L.) provenances cover broad ecological amplitudes. In a greenhouse study, we investigated the impact of drought stress and rewetting on gas exchange for three provenances (Italy: Emilia Romagna; Spain: Alto Ebro; Germany: East-German lowlands) of 2-year old Scots pine seedlings. CO 2 , water vapour and isoprenoid exchange of stressed and control trees were quantified with a four-chamber dynamic-enclosure system in the controlled environment of a climate chamber. The three provenances showed distinct isoprenoid emission patterns and were classified into a non-Δ 3 -carene, with either high α-/β-pinene or β-myrcene fraction, and a Δ 3 -carene dominated type. Isoprenoid emission rates, net-photosynthesis and transpiration were reduced during summer drought stress and significantly recovered after rewetting. A seasonal increase of isoprenoid emission rates towards autumn was observed for all control groups. Compared with the German provenance, the Spanish and Italian provenances revealed higher isoprenoid emission rates and more plastic responses to drought stress and seasonal development, which points to a local adaptation to climate. As a result of drought, net carbon uptake and transpiration of trees was reduced, but recovered after rewetting. We conclude from our study that Scots pine isoprenoid emission is more variable than expected and sensitive to drought periods, likely impacting regional air chemistry. Thus, a provenance-specific emission assessment accounting for reduced emission during prolonged (summer) drought is recommend for setting up biogenic volatile organic compound emission inventories used in air quality models. © The Author 2016. Published by Oxford University Press.

The functional readthrough extension of malate dehydrogenase reveals a modification of the genetic code

PubMed Central

Hofhuis, Julia; Schueren, Fabian; Nötzel, Christopher; Lingner, Thomas; Gärtner, Jutta; Jahn, Olaf

2016-01-01

Translational readthrough gives rise to C-terminally extended proteins, thereby providing the cell with new protein isoforms. These may have different properties from the parental proteins if the extensions contain functional domains. While for most genes amino acid incorporation at the stop codon is far lower than 0.1%, about 4% of malate dehydrogenase (MDH1) is physiologically extended by translational readthrough and the actual ratio of MDH1x (extended protein) to ‘normal' MDH1 is dependent on the cell type. In human cells, arginine and tryptophan are co-encoded by the MDH1x UGA stop codon. Readthrough is controlled by the 7-nucleotide high-readthrough stop codon context without contribution of the subsequent 50 nucleotides encoding the extension. All vertebrate MDH1x is directed to peroxisomes via a hidden peroxisomal targeting signal (PTS) in the readthrough extension, which is more highly conserved than the extension of lactate dehydrogenase B. The hidden PTS of non-mammalian MDH1x evolved to be more efficient than the PTS of mammalian MDH1x. These results provide insight into the genetic and functional co-evolution of these dually localized dehydrogenases. PMID:27881739

Genome-wide analysis of codon usage bias in four sequenced cotton species.

PubMed

Wang, Liyuan; Xing, Huixian; Yuan, Yanchao; Wang, Xianlin; Saeed, Muhammad; Tao, Jincai; Feng, Wei; Zhang, Guihua; Song, Xianliang; Sun, Xuezhen

2018-01-01

Codon usage bias (CUB) is an important evolutionary feature in a genome which provides important information for studying organism evolution, gene function and exogenous gene expression. The CUB and its shaping factors in the nuclear genomes of four sequenced cotton species, G. arboreum (A2), G. raimondii (D5), G. hirsutum (AD1) and G. barbadense (AD2) were analyzed in the present study. The effective number of codons (ENC) analysis showed the CUB was weak in these four species and the four subgenomes of the two tetraploids. Codon composition analysis revealed these four species preferred to use pyrimidine-rich codons more frequently than purine-rich codons. Correlation analysis indicated that the base content at the third position of codons affect the degree of codon preference. PR2-bias plot and ENC-plot analyses revealed that the CUB patterns in these genomes and subgenomes were influenced by combined effects of translational selection, directional mutation and other factors. The translational selection (P2) analysis results, together with the non-significant correlation between GC12 and GC3, further revealed that translational selection played the dominant role over mutation pressure in the codon usage bias. Through relative synonymous codon usage (RSCU) analysis, we detected 25 high frequency codons preferred to end with T or A, and 31 low frequency codons inclined to end with C or G in these four species and four subgenomes. Finally, 19 to 26 optimal codons with 19 common ones were determined for each species and subgenomes, which preferred to end with A or T. We concluded that the codon usage bias was weak and the translation selection was the main shaping factor in nuclear genes of these four cotton genomes and four subgenomes.

Discovery of silicified lacustrine micro-fossils and stromatolites: Triassic-Jurassic Fundy Group, Nova Scotia

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

Cameron, B.

A unique assemblage of silicified invertebrate and algal fresh-water lake fossils has been discovered in the Scots Bay Formation at the top of the Triassic-Jurassic Fundy Group of the Fundy Basin in Nova Scotia. This is important because the basins of the eastern North American Triassic-Jurassic rift system have not yielded many invertebrate and algal fossils. These new finds will contribute significantly to evolutionary, paleoecological and biostratigraphic studies of fresh-water Mesozoic deposits. Silicified fossils have been extracted from chert-bearing, mixed carbonate and siliciclastic lithologies. They include ostracodes, gastropods, rare bivalves, charaphytes (algae), stromatolites, and chert nodules cored with well-preserved woodymore » tissues of tree trunks. Possible algal filaments occur in the silicified stromatolites. This association of charaphytes, ostracodes, microscopic gastropods and stromatolites is found in carbonate lakes today. The Scots Bay Formation is probably a near-shore carbonate facies of the more widespread silicilastic lacustrine McCoy Brook Formation. The gastropods and ostracodes, studied by SEM, indicate a Jurassic age for the Scots bay Formation, confirming speculations based on other data.« less

When one disease is not enough: succinyl-CoA: 3-oxoacid coenzyme A transferase (SCOT) deficiency due to a novel mutation in OXCT1 in an infant with known phenylketonuria.

PubMed

Schwade, Jan-Niclas; Endmann, Matthias; Hofmann, Thomas; Rust, Stephan; Sass, Jörn Oliver; Rutsch, Frank

2017-10-26

A 9-month-old Turkish girl was admitted several times within 3 months to the hospital in reduced general condition and with extreme tachypnea. The patient had been diagnosed with phenylketonuria (PKU) in newborn screening and has been treated with a low phenylalanine diet and amino acid supplements. Each time an unexplained pronounced metabolic acidosis was noted, and the child was treated with sodium-bicarbonate and glucose-electrolyte infusions. The acidosis with only slightly abnormal glucose, normal lactate levels and pronounced ketonuria suggested a defect in ketone body utilization. Succinyl-CoA: 3-oxoacid CoA transferase (SCOT) enzyme activity was low in patient's fibroblasts. Mutation analysis of the corresponding OXCT1 gene revealed that the patient was a homozygous carrier of the mutation c.1523T>C (p.V508A). We conclude that SCOT deficiency should be considered in the differential diagnosis in patients with recurrent metabolic acidotic episodes, even if they are already known to have a metabolic disease unrelated to this.

Effect of codon optimization and subcellular targeting on Toxoplasma gondii antigen SAG1 expression in tobacco leaves to use in subcutaneous and oral immunization in mice.

PubMed

Laguía-Becher, Melina; Martín, Valentina; Kraemer, Mauricio; Corigliano, Mariana; Yacono, María L; Goldman, Alejandra; Clemente, Marina

2010-07-15

Codon optimization and subcellular targeting were studied with the aim to increase the expression levels of the SAG178-322 antigen of Toxoplasma gondii in tobacco leaves. The expression of the tobacco-optimized and native versions of the SAG1 gene was explored by transient expression from the Agrobacterium tumefaciens binary expression vector, which allows targeting the recombinant protein to the endoplasmic reticulum (ER) and the apoplast. Finally, mice were subcutaneously and orally immunized with leaf extracts-SAG1 and the strategy of prime boost with rSAG1 expressed in Escherichia coli was used to optimize the oral immunization with leaf extracts-SAG1. Leaves agroinfiltrated with an unmodified SAG1 gene accumulated 5- to 10-fold more than leaves agroinfiltrated with a codon-optimized SAG1 gene. ER localization allowed the accumulation of higher levels of native SAG1. However, no significant differences were observed between the mRNA accumulations of the different versions of SAG1. Subcutaneous immunization with leaf extracts-SAG1 (SAG1) protected mice against an oral challenge with a non-lethal cyst dose, and this effect could be associated with the secretion of significant levels of IFN-gamma. The protection was increased when mice were ID boosted with rSAG1 (SAG1+boost). This group elicited a significant Th1 humoral and cellular immune response characterized by high levels of IFN-gamma. In an oral immunization assay, the SAG1+boost group showed a significantly lower brain cyst burden compared to the rest of the groups. Transient agroinfiltration was useful for the expression of all of the recombinant proteins tested. Our results support the usefulness of endoplasmic reticulum signal peptides in enhancing the production of recombinant proteins meant for use as vaccines. The results showed that this plant-produced protein has potential for use as vaccine and provides a potential means for protecting humans and animals against toxoplasmosis.

Codon 219 polymorphism of PRNP in healthy caucasians and Creutzfeldt-Jakob disease patients

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

Petraroli, R.; Pocchiari, M.

1996-04-01

A number of point and insert mutations of the PrP gene (PRNP) have been linked to familial Creutzfeldt-Jakob disease (CJD) and Gerstmann-Straussler-Scheinker disease (GSS). Moreover, the methionine/valine homozygosity at the polymorphic codon 129 of PRNP may cause a predisposition to sporadic and iatrogenic CJD or may control the age at onset of familial cases carrying either the 144-bp insertion or codon 178, codon 198, and codon 210 pathogenic mutations in PRNP. In addition, the association of methionine or valine at codon 129 and the point mutation at codon 178 on the same allele seem to play an important role inmore » determining either fatal familial insomnia or CJD. However, it is noteworthy that a relationship between codon 129 polymorphism and accelerated pathogenesis (early age at onset or shorter duration of the disease) has not been seen in familial CJD patients with codon 200 mutation or in GSS patients with codon 102 mutation, arguing that other, as yet unidentified, gene products or environmental factors, or both, may influence the clinical expression of these diseases. 17 refs.« less

Emergent rules for codon choice elucidated by editing rare arginine codons in Escherichia coli

PubMed Central

Napolitano, Michael G.; Landon, Matthieu; Gregg, Christopher J.; Lajoie, Marc J.; Govindarajan, Lakshmi; Mosberg, Joshua A.; Kuznetsov, Gleb; Goodman, Daniel B.; Vargas-Rodriguez, Oscar; Isaacs, Farren J.; Söll, Dieter; Church, George M.

2016-01-01

The degeneracy of the genetic code allows nucleic acids to encode amino acid identity as well as noncoding information for gene regulation and genome maintenance. The rare arginine codons AGA and AGG (AGR) present a case study in codon choice, with AGRs encoding important transcriptional and translational properties distinct from the other synonymous alternatives (CGN). We created a strain of Escherichia coli with all 123 instances of AGR codons removed from all essential genes. We readily replaced 110 AGR codons with the synonymous CGU codons, but the remaining 13 “recalcitrant” AGRs required diversification to identify viable alternatives. Successful replacement codons tended to conserve local ribosomal binding site-like motifs and local mRNA secondary structure, sometimes at the expense of amino acid identity. Based on these observations, we empirically defined metrics for a multidimensional “safe replacement zone” (SRZ) within which alternative codons are more likely to be viable. To evaluate synonymous and nonsynonymous alternatives to essential AGRs further, we implemented a CRISPR/Cas9-based method to deplete a diversified population of a wild-type allele, allowing us to evaluate exhaustively the fitness impact of all 64 codon alternatives. Using this method, we confirmed the relevance of the SRZ by tracking codon fitness over time in 14 different genes, finding that codons that fall outside the SRZ are rapidly depleted from a growing population. Our unbiased and systematic strategy for identifying unpredicted design flaws in synthetic genomes and for elucidating rules governing codon choice will be crucial for designing genomes exhibiting radically altered genetic codes. PMID:27601680

Exploring codon context bias for synthetic gene design of a thermostable invertase in Escherichia coli.

PubMed

Pek, Han Bin; Klement, Maximilian; Ang, Kok Siong; Chung, Bevan Kai-Sheng; Ow, Dave Siak-Wei; Lee, Dong-Yup

2015-01-01

Various isoforms of invertases from prokaryotes, fungi, and higher plants has been expressed in Escherichia coli, and codon optimisation is a widely-adopted strategy for improvement of heterologous enzyme expression. Successful synthetic gene design for recombinant protein expression can be done by matching its translational elongation rate against heterologous host organisms via codon optimization. Amongst the various design parameters considered for the gene synthesis, codon context bias has been relatively overlooked compared to individual codon usage which is commonly adopted in most of codon optimization tools. In addition, matching the rates of transcription and translation based on secondary structure may lead to enhanced protein folding. In this study, we evaluated codon context fitness as design criterion for improving the expression of thermostable invertase from Thermotoga maritima in Escherichia coli and explored the relevance of secondary structure regions for folding and expression. We designed three coding sequences by using (1) a commercial vendor optimized gene algorithm, (2) codon context for the whole gene, and (3) codon context based on the secondary structure regions. Then, the codon optimized sequences were transformed and expressed in E. coli. From the resultant enzyme activities and protein yield data, codon context fitness proved to have the highest activity as compared to the wild-type control and other criteria while secondary structure-based strategy is comparable to the control. Codon context bias was shown to be a relevant parameter for enhancing enzyme production in Escherichia coli by codon optimization. Thus, we can effectively design synthetic genes within heterologous host organisms using this criterion. Copyright © 2015 Elsevier Inc. All rights reserved.

The Effect of an Alternate Start Codon on Heterologous Expression of a PhoA Fusion Protein in Mycoplasma gallisepticum

PubMed Central

Panicker, Indu S.; Browning, Glenn F.; Markham, Philip F.

2015-01-01

While the genomes of many Mycoplasma species have been sequenced, there are no collated data on translational start codon usage, and the effects of alternate start codons on gene expression have not been studied. Analysis of the annotated genomes found that ATG was the most prevalent translational start codon among Mycoplasma spp. However in Mycoplasma gallisepticum a GTG start codon is commonly used in the vlhA multigene family, which encodes a highly abundant, phase variable lipoprotein adhesin. Therefore, the effect of this alternate start codon on expression of a reporter PhoA lipoprotein was examined in M. gallisepticum. Mutation of the start codon from ATG to GTG resulted in a 2.5 fold reduction in the level of transcription of the phoA reporter, but the level of PhoA activity in the transformants containing phoA with a GTG start codon was only 63% of that of the transformants with a phoA with an ATG start codon, suggesting that GTG was a more efficient translational initiation codon. The effect of swapping the translational start codon in phoA reporter gene expression was less in M. gallisepticum than has been seen previously in Escherichia coli or Bacillus subtilis, suggesting the process of translational initiation in mycoplasmas may have some significant differences from those used in other bacteria. This is the first study of translational start codon usage in mycoplasmas and the impact of the use of an alternate start codon on expression in these bacteria. PMID:26010086

Zebrafish Model of NF1 for Structure-Function Analysis, Mechanisms of Glial Tumorigenesis, and Chemical Biology

DTIC Science & Technology

2015-08-01

Meng1,‡, Jimann Shin2, Arun Padmanabhan3, Daniel Cifuentes4, Antonio J. Giraldez4, A. Thomas Look2, Jonathan A. Epstein3, Nathan D. Lawson1,§ and Scot ...Geurts, A. M., Cost, G. J., Freyvert, Y., Zeitler, B., Miller, J. C., Choi, V. M., Jenkins, S. S., Wood , A., Cui, X., Meng, X. et al. (2009). Knockout...de Groh2,‡, Jeong-Soo Lee1,§, Sam Haidar3, Suzanne Dahlberg1, Feng Guo1, Shuning He1, Marc A. Wolman2, Michael Granato2, Nathan D. Lawson4, Scot A

Elemental analyses of pine bark and wood in an environmental study.

PubMed

Saarela, K-E; Harju, L; Rajander, J; Lill, J-O; Heselius, S-J; Lindroos, A; Mattsson, K

2005-05-01

Bark and wood samples were taken from the same individuals of Scots pine (Pinus sylvestris L.) from a polluted area close to a Cu-Ni smelter in Harjavalta and from some relatively unpolluted areas in western Finland. The samples were analysed by thick-target particle induced X-ray emission (PIXE) after preconcentration by dry ashing at 550 degrees C. The elemental contents of pine bark and wood were compared to study the impact of heavy metal pollution on pine trees. By comparison of the elemental contents in ashes of bark and wood, a normalisation was obtained. For the relatively clean areas, the ratios of the concentration in bark ash to the concentration in wood ash for different elements were close to 1. This means that the ashes of Scots Pine wood and bark have quite similar elemental composition. For the samples from the polluted area the mean concentration ratios for some heavy metals were elevated (13-28), reflecting the effect of direct atmospheric contamination. The metal contents in the ashes of pine bark and wood were also compared to recommendations for ashes to be recycled back to the forest environment. Bark from areas close to emission sources of heavy metal pollution should be considered with caution if aiming at recycling the ash. Burning of bark fuel of pine grown within 6 km of the Cu-Ni smelter is shown to generate ashes with high levels of Cu, Ni as well as Cd, As and Pb.

Amino acid repeats avert mRNA folding through conservative substitutions and synonymous codons, regardless of codon bias.

PubMed

Barik, Sailen

2017-12-01

A significant number of proteins in all living species contains amino acid repeats (AARs) of various lengths and compositions, many of which play important roles in protein structure and function. Here, I have surveyed select homopolymeric single [(A)n] and double [(AB)n] AARs in the human proteome. A close examination of their codon pattern and analysis of RNA structure propensity led to the following set of empirical rules: (1) One class of amino acid repeats (Class I) uses a mixture of synonymous codons, some of which approximate the codon bias ratio in the overall human proteome; (2) The second class (Class II) disregards the codon bias ratio, and appears to have originated by simple repetition of the same codon (or just a few codons); and finally, (3) In all AARs (including Class I, Class II, and the in-betweens), the codons are chosen in a manner that precludes the formation of RNA secondary structure. It appears that the AAR genes have evolved by orchestrating a balance between codon usage and mRNA secondary structure. The insights gained here should provide a better understanding of AAR evolution and may assist in designing synthetic genes.

Complex codon usage pattern and compositional features of retroviruses.

PubMed

RoyChoudhury, Sourav; Mukherjee, Debaprasad

2013-01-01

Retroviruses infect a wide range of organisms including humans. Among them, HIV-1, which causes AIDS, has now become a major threat for world health. Some of these viruses are also potential gene transfer vectors. In this study, the patterns of synonymous codon usage in retroviruses have been studied through multivariate statistical methods on ORFs sequences from the available 56 retroviruses. The principal determinant for evolution of the codon usage pattern in retroviruses seemed to be the compositional constraints, while selection for translation of the viral genes plays a secondary role. This was further supported by multivariate analysis on relative synonymous codon usage. Thus, it seems that mutational bias might have dominated role over translational selection in shaping the codon usage of retroviruses. Codon adaptation index was used to identify translationally optimal codons among genes from retroviruses. The comparative analysis of the preferred and optimal codons among different retroviral groups revealed that four codons GAA, AAA, AGA, and GGA were significantly more frequent in most of the retroviral genes inspite of some differences. Cluster analysis also revealed that phylogenetically related groups of retroviruses have probably evolved their codon usage in a concerted manner under the influence of their nucleotide composition.

Synonymous codon usage patterns in different parasitic platyhelminth mitochondrial genomes.

PubMed

Chen, L; Yang, D Y; Liu, T F; Nong, X; Huang, X; Xie, Y; Fu, Y; Zheng, W P; Zhang, R H; Wu, X H; Gu, X B; Wang, S X; Peng, X R; Yang, G Y

2013-02-27

We analyzed synonymous codon usage patterns of the mitochondrial genomes of 43 parasitic platyhelminth species. The relative synonymous codon usage, the effective number of codons (NC) and the frequency of G+C at the third synonymously variable coding position were calculated. Correspondence analysis was used to determine the major variation trends shaping the codon usage patterns. Among the mitochondrial genomes of 19 trematode species, the GC content of third codon positions varied from 0.151 to 0.592, with a mean of 0.295 ± 0.116. In cestodes, the mean GC content of third codon positions was 0.254 ± 0.044. A comparison of the nucleotide composition at 4-fold synonymous sites revealed that, on average, there was a greater abundance of codons ending on U (51.9%) or A (22.7%) than on C (6.3%) or G (19.14%). Twenty-two codons, including UUU, UUA and UUG, were frequently used. In the NC-plot, most of points were distributed well below or around the expected NC curve. In addition to compositional constraints, the degree of hydrophobicity and the aromatic amino acids also influenced codon usage in the mitochondrial genomes of these 43 parasitic platyhelminth species.

The intracellular Scots pine shoot symbiont Methylobacterium extorquens DSM13060 aggregates around the host nucleus and encodes eukaryote-like proteins.

PubMed

Koskimäki, Janne J; Pirttilä, Anna Maria; Ihantola, Emmi-Leena; Halonen, Outi; Frank, A Carolin

2015-03-24

Endophytes are microbes that inhabit plant tissues without any apparent signs of infection, often fundamentally altering plant phenotypes. While endophytes are typically studied in plant roots, where they colonize the apoplast or dead cells, Methylobacterium extorquens strain DSM13060 is a facultatively intracellular symbiont of the meristematic cells of Scots pine (Pinus sylvestris L.) shoot tips. The bacterium promotes host growth and development without the production of known plant growth-stimulating factors. Our objective was to examine intracellular colonization by M. extorquens DSM13060 of Scots pine and sequence its genome to identify novel molecular mechanisms potentially involved in intracellular colonization and plant growth promotion. Reporter construct analysis of known growth promotion genes demonstrated that these were only weakly active inside the plant or not expressed at all. We found that bacterial cells accumulate near the nucleus in intact, living pine cells, pointing to host nuclear processes as the target of the symbiont's activity. Genome analysis identified a set of eukaryote-like functions that are common as effectors in intracellular bacterial pathogens, supporting the notion of intracellular bacterial activity. These include ankyrin repeats, transcription factors, and host-defense silencing functions and may be secreted by a recently imported type IV secretion system. Potential factors involved in host growth include three copies of phospholipase A2, an enzyme that is rare in bacteria but implicated in a range of plant cellular processes, and proteins putatively involved in gibberellin biosynthesis. Our results describe a novel endophytic niche and create a foundation for postgenomic studies of a symbiosis with potential applications in forestry and agriculture. All multicellular eukaryotes host communities of essential microbes, but most of these interactions are still poorly understood. In plants, bacterial endophytes are found inside all tissues. M. extorquens DSM13060 occupies an unusual niche inside cells of the dividing shoot tissues of a pine and stimulates seedling growth without producing cytokinin, auxin, or other plant hormones commonly synthesized by plant-associated bacteria. Here, we tracked the bacteria using a fluorescent tag and confocal laser scanning microscopy and found that they localize near the nucleus of the plant cell. This prompted us to sequence the genome and identify proteins that may affect host growth by targeting processes in the host cytoplasm and nucleus. We found many novel genes whose products may modulate plant processes from within the plant cell. Our results open up new avenues to better understand how bacteria assist in plant growth, with broad implications for plant science, forestry, and agriculture. Copyright © 2015 Koskimäki et al.

Essentiality, conservation, evolutionary pressure and codon bias in bacterial genomes.

PubMed

Dilucca, Maddalena; Cimini, Giulio; Giansanti, Andrea

2018-07-15

Essential genes constitute the core of genes which cannot be mutated too much nor lost along the evolutionary history of a species. Natural selection is expected to be stricter on essential genes and on conserved (highly shared) genes, than on genes that are either nonessential or peculiar to a single or a few species. In order to further assess this expectation, we study here how essentiality of a gene is connected with its degree of conservation among several unrelated bacterial species, each one characterised by its own codon usage bias. Confirming previous results on E. coli, we show the existence of a universal exponential relation between gene essentiality and conservation in bacteria. Moreover, we show that, within each bacterial genome, there are at least two groups of functionally distinct genes, characterised by different levels of conservation and codon bias: i) a core of essential genes, mainly related to cellular information processing; ii) a set of less conserved nonessential genes with prevalent functions related to metabolism. In particular, the genes in the first group are more retained among species, are subject to a stronger purifying conservative selection and display a more limited repertoire of synonymous codons. The core of essential genes is close to the minimal bacterial genome, which is in the focus of recent studies in synthetic biology, though we confirm that orthologs of genes that are essential in one species are not necessarily essential in other species. We also list a set of highly shared genes which, reasonably, could constitute a reservoir of targets for new anti-microbial drugs. Copyright © 2018 Elsevier B.V. All rights reserved.

Codon usage bias: causative factors, quantification methods and genome-wide patterns: with emphasis on insect genomes.

PubMed

Behura, Susanta K; Severson, David W

2013-02-01

Codon usage bias refers to the phenomenon where specific codons are used more often than other synonymous codons during translation of genes, the extent of which varies within and among species. Molecular evolutionary investigations suggest that codon bias is manifested as a result of balance between mutational and translational selection of such genes and that this phenomenon is widespread across species and may contribute to genome evolution in a significant manner. With the advent of whole-genome sequencing of numerous species, both prokaryotes and eukaryotes, genome-wide patterns of codon bias are emerging in different organisms. Various factors such as expression level, GC content, recombination rates, RNA stability, codon position, gene length and others (including environmental stress and population size) can influence codon usage bias within and among species. Moreover, there has been a continuous quest towards developing new concepts and tools to measure the extent of codon usage bias of genes. In this review, we outline the fundamental concepts of evolution of the genetic code, discuss various factors that may influence biased usage of synonymous codons and then outline different principles and methods of measurement of codon usage bias. Finally, we discuss selected studies performed using whole-genome sequences of different insect species to show how codon bias patterns vary within and among genomes. We conclude with generalized remarks on specific emerging aspects of codon bias studies and highlight the recent explosion of genome-sequencing efforts on arthropods (such as twelve Drosophila species, species of ants, honeybee, Nasonia and Anopheles mosquitoes as well as the recent launch of a genome-sequencing project involving 5000 insects and other arthropods) that may help us to understand better the evolution of codon bias and its biological significance. © 2012 The Authors. Biological Reviews © 2012 Cambridge Philosophical Society.

Selective forces and mutational biases drive stop codon usage in the human genome: a comparison with sense codon usage.

PubMed

Trotta, Edoardo

2016-05-17

The three stop codons UAA, UAG, and UGA signal the termination of mRNA translation. As a result of a mechanism that is not adequately understood, they are normally used with unequal frequencies. In this work, we showed that selective forces and mutational biases drive stop codon usage in the human genome. We found that, in respect to sense codons, stop codon usage was affected by stronger selective forces but was less influenced by neutral mutational biases. UGA is the most frequent termination codon in human genome. However, UAA was the preferred stop codon in genes with high breadth of expression, high level of expression, AT-rich coding sequences, housekeeping functions, and in gene ontology categories with the largest deviation from expected stop codon usage. Selective forces associated with the breadth and the level of expression favoured AT-rich sequences in the mRNA region including the stop site and its proximal 3'-UTR, but acted with scarce effects on sense codons, generating two regions, upstream and downstream of the stop codon, with strongly different base composition. By favouring low levels of GC-content, selection promoted labile local secondary structures at the stop site and its proximal 3'-UTR. The compositional and structural context favoured by selection was surprisingly emphasized in the class of ribosomal proteins and was consistent with sequence elements that increase the efficiency of translational termination. Stop codons were also heterogeneously distributed among chromosomes by a mechanism that was strongly correlated with the GC-content of coding sequences. In human genome, the nucleotide composition and the thermodynamic stability of stop codon site and its proximal 3'-UTR are correlated with the GC-content of coding sequences and with the breadth and the level of gene expression. In highly expressed genes stop codon usage is compositionally and structurally consistent with highly efficient translation termination signals.

Pandemic influenza A virus codon usage revisited: biases, adaptation and implications for vaccine strain development

PubMed Central

2012-01-01

Background Influenza A virus (IAV) is a member of the family Orthomyxoviridae and contains eight segments of a single-stranded RNA genome with negative polarity. The first influenza pandemic of this century was declared in April of 2009, with the emergence of a novel H1N1 IAV strain (H1N1pdm) in Mexico and USA. Understanding the extent and causes of biases in codon usage is essential to the understanding of viral evolution. A comprehensive study to investigate the effect of selection pressure imposed by the human host on the codon usage of an emerging, pandemic IAV strain and the trends in viral codon usage involved over the pandemic time period is much needed. Results We performed a comprehensive codon usage analysis of 310 IAV strains from the pandemic of 2009. Highly biased codon usage for Ala, Arg, Pro, Thr and Ser were found. Codon usage is strongly influenced by underlying biases in base composition. When correspondence analysis (COA) on relative synonymous codon usage (RSCU) is applied, the distribution of IAV ORFs in the plane defined by the first two major dimensional factors showed that different strains are located at different places, suggesting that IAV codon usage also reflects an evolutionary process. Conclusions A general association between codon usage bias, base composition and poor adaptation of the virus to the respective host tRNA pool, suggests that mutational pressure is the main force shaping H1N1 pdm IAV codon usage. A dynamic process is observed in the variation of codon usage of the strains enrolled in these studies. These results suggest a balance of mutational bias and natural selection, which allow the virus to explore and re-adapt its codon usage to different environments. Recoding of IAV taking into account codon bias, base composition and adaptation to host tRNA may provide important clues to develop new and appropriate vaccines. PMID:23134595

Evaluating Sense Codon Reassignment with a Simple Fluorescence Screen.

PubMed

Biddle, Wil; Schmitt, Margaret A; Fisk, John D

2015-12-22

Understanding the interactions that drive the fidelity of the genetic code and the limits to which modifications can be made without breaking the translational system has practical implications for understanding the molecular mechanisms of evolution as well as expanding the set of encodable amino acids, particularly those with chemistries not provided by Nature. Because 61 sense codons encode 20 amino acids, reassigning the meaning of sense codons provides an avenue for biosynthetic modification of proteins, furthering both fundamental and applied biochemical research. We developed a simple screen that exploits the absolute requirement for fluorescence of an active site tyrosine in green fluorescent protein (GFP) to probe the pliability of the degeneracy of the genetic code. Our screen monitors the restoration of the fluorophore of GFP by incorporation of a tyrosine in response to a sense codon typically assigned another meaning in the genetic code. We evaluated sense codon reassignment at four of the 21 sense codons read through wobble interactions in Escherichia coli using the Methanocaldococcus jannaschii orthogonal tRNA/aminoacyl tRNA synthetase pair originally developed and commonly used for amber stop codon suppression. By changing only the anticodon of the orthogonal tRNA, we achieved sense codon reassignment efficiencies between 1% (Phe UUU) and 6% (Lys AAG). Each of the orthogonal tRNAs preferentially decoded the codon traditionally read via a wobble interaction in E. coli with the exception of the orthogonal tRNA with an AUG anticodon, which incorporated tyrosine in response to both the His CAU and His CAC codons with approximately equal frequencies. We applied our screen in a high-throughput manner to evaluate a 10(9)-member combined tRNA/aminoacyl tRNA synthetase library to identify improved sense codon reassigning variants for the Lys AAG codon. A single rapid screen with the ability to broadly evaluate reassignable codons will facilitate identification and improvement of the combinations of sense codons and orthogonal pairs that display efficient reassignment.

Increase of apatite dissolution rate by Scots pine roots associated or not with Burkholderia glathei PML1(12)Rp in open-system flow microcosms

NASA Astrophysics Data System (ADS)

Calvaruso, Christophe; Turpault, Marie-Pierre; Frey-Klett, Pascale; Uroz, Stéphane; Pierret, Marie-Claire; Tosheva, Zornitza; Kies, Antoine

2013-04-01

The release of nutritive elements through apatite dissolution represents the main source of phosphorus, calcium, and several micronutrients (e.g., Zn, Cu) for organisms in non-fertilized forest ecosystems. The aim of this study was to quantify, for the first time, the dissolution rate of apatite grains by tree roots that were or were not associated with a mineral weathering bacterial strain, and by various acids known to be produced by tree roots and soil bacterial strains in open-system flow microcosms. In addition, we explored whether the mobilization of trace elements (including rare earth elements) upon apatite dissolution was affected by the presence of trees and associated microorganisms. The dissolution rate of apatite by Scots pine plants that were or were not inoculated with the strain Burkholderia glathei PML1(12)Rp, and by inorganic (nitric) and organic (citric, oxalic and gluconic) acids at pH 5.5, 4.8, 3.8, 3.5, 3.0, and 2.0 was monitored in two controlled experiments: "plant-bacteria interaction" and "inorganic and organic acids". Analyses of the outlet solutions in the "plant-bacteria interaction" experiment showed that Scots pine roots and B. glathei PML1(12)Rp produced protons and organic acids such as gluconate, oxalate, acetate, and lactate. The weathering budget calculation revealed that Scots pines (with or without PML1(12)Rp) significantly increased (factor > 10) the release of Ca, P, As, Sr, Zn, U, Y, and rare earth elements such as Ce, La, Nd from apatite, compared to control abiotic treatment. Scanning electron microscopy observation confirmed traces of apatite dissolution in contact of roots. Most dissolved elements were taken up by Scots pine roots, i.e., approximately 50% of Ca, 70% of P, 30% of As, 70% of Sr, 90% of Zn, and 100% of U, Y, and rare earth elements. Interestingly, no significant additional effect due to the bacterial strain PML1(12)Rp on apatite dissolution and Scots pine nutrition and growth was observed. The "inorganic and organic acids" experiment demonstrated that the apatite dissolution efficacy of organic acids was higher than for the inorganic acid and varied in function of the acids: oxalic acid > citric acid > gluconic acid > nitric acid for pH ⩽3.5. In addition, apatite dissolution increased with increasing acidity for each acid. Only oxalic acid generated non-stoichiometric release of calcium and phosphorus from apatite in the solution at pH ⩽3.5, due to the precipitation of Ca-oxalate crystals at apatite surfaces. Comparison of the experiments revealed that the apatite dissolution rate by Scots pines supplied with nutritive solution at pH 5.5 reached 2.0 × 10-13 mol cm-2 s-1 and was equivalent to rates with nitric acid at pH 3.2, gluconic acid at pH 3.5, citric acid at pH 3.7, and oxalic acid at pH 3.8. Altogether our results highlight that, through the production of weathering agents, notably protons and organic acids, tree roots and root-associated microorganisms are able to significantly increase the release of macro- and micro-nutrients from apatite, thus maintaining high-nutrient conditions to support their growth.

Revelation of Influencing Factors in Overall Codon Usage Bias of Equine Influenza Viruses

PubMed Central

Bhatia, Sandeep; Sood, Richa; Selvaraj, Pavulraj

2016-01-01

Equine influenza viruses (EIVs) of H3N8 subtype are culprits of severe acute respiratory infections in horses, and are still responsible for significant outbreaks worldwide. Adaptability of influenza viruses to a particular host is significantly influenced by their codon usage preference, due to an absolute dependence on the host cellular machinery for their replication. In the present study, we analyzed genome-wide codon usage patterns in 92 EIV strains, including both H3N8 and H7N7 subtypes by computing several codon usage indices and applying multivariate statistical methods. Relative synonymous codon usage (RSCU) analysis disclosed bias of preferred synonymous codons towards A/U-ended codons. The overall codon usage bias in EIVs was slightly lower, and mainly affected by the nucleotide compositional constraints as inferred from the RSCU and effective number of codon (ENc) analysis. Our data suggested that codon usage pattern in EIVs is governed by the interplay of mutation pressure, natural selection from its hosts and undefined factors. The H7N7 subtype was found less fit to its host (horse) in comparison to H3N8, by possessing higher codon bias, lower mutation pressure and much less adaptation to tRNA pool of equine cells. To the best of our knowledge, this is the first report describing the codon usage analysis of the complete genomes of EIVs. The outcome of our study is likely to enhance our understanding of factors involved in viral adaptation, evolution, and fitness towards their hosts. PMID:27119730

Response of central Siberian Scots pine to soil water deficit and long-term trends in atmospheric CO2 concentration

NASA Astrophysics Data System (ADS)

Arneth, A.; Lloyd, J.; Šantrůčková, H.; Bird, M.; Grigoryev, S.; Kalaschnikov, Y. N.; Gleixner, G.; Schulze, E.-D.

2002-01-01

Twenty tree ring 13C / 12C ratio chronologies from Pinus sylvestris (Scots pine) trees were determined from five locations sampled along the Yenisei River, spaced over a total distance of ~1000 km between the cities of Turuhansk (66°N) and Krasnoyarsk (56°N). The transect covered the major part of the natural distribution of Scots pine in the region with median growing season temperatures and precipitation varying from 12.2°C and 218 mm to 14.0°C and 278 mm for Turuhansk and Krasnoyarsk, respectively. A key focus of the study was to investigate the effects of variations in temperature, precipitation, and atmospheric CO2 concentration on long- and short-term variation in photosynthetic 13C discrimination during photosynthesis and the marginal cost of tree water use, as reflected in the differences in the historical records of the 13C / 12C ratio in wood cellulose compared to that of the atmosphere (Δ13Cc). In 17 of the 20 samples, trees Δ13Cc has declined during the last 150 years, particularly so during the second half of the twentieth century. Using a model of stomatal behaviour combined with a process-based photosynthesis model, we deduce that this trend indicates a long-term decrease in canopy stomatal conductance, probably in response to increasing atmospheric CO2 concentrations. This response being observed for most trees along the transect is suggestive of widespread decreases in Δ13Cc and increased water use efficiency for Scots pine in central Siberia over the last century. Overlying short-term variations in Δ13Cc were also accounted for by the model and were related to variations in growing season soil water deficit and atmospheric humidity.

Tree-growth analyses to estimate tree species' drought tolerance.

PubMed

Eilmann, Britta; Rigling, Andreas

2012-02-01

Climate change is challenging forestry management and practices. Among other things, tree species with the ability to cope with more extreme climate conditions have to be identified. However, while environmental factors may severely limit tree growth or even cause tree death, assessing a tree species' potential for surviving future aggravated environmental conditions is rather demanding. The aim of this study was to find a tree-ring-based method suitable for identifying very drought-tolerant species, particularly potential substitute species for Scots pine (Pinus sylvestris L.) in Valais. In this inner-Alpine valley, Scots pine used to be the dominating species for dry forests, but today it suffers from high drought-induced mortality. We investigate the growth response of two native tree species, Scots pine and European larch (Larix decidua Mill.), and two non-native species, black pine (Pinus nigra Arnold) and Douglas fir (Pseudotsuga menziesii Mirb. var. menziesii), to drought. This involved analysing how the radial increment of these species responded to increasing water shortage (abandonment of irrigation) and to increasingly frequent drought years. Black pine and Douglas fir are able to cope with drought better than Scots pine and larch, as they show relatively high radial growth even after irrigation has been stopped and a plastic growth response to drought years. European larch does not seem to be able to cope with these dry conditions as it lacks the ability to recover from drought years. The analysis of trees' short-term response to extreme climate events seems to be the most promising and suitable method for detecting how tolerant a tree species is towards drought. However, combining all the methods used in this study provides a complete picture of how water shortage could limit species.

Absence of opioid stress-induced analgesia in mice lacking beta-endorphin by site-directed mutagenesis.

PubMed

Rubinstein, M; Mogil, J S; Japón, M; Chan, E C; Allen, R G; Low, M J

1996-04-30

A physiological role for beta-endorphin in endogenous pain inhibition was investigated by targeted mutagenesis of the proopiomelanocortin gene in mouse embryonic stem cells. The tyrosine codon at position 179 of the proopiomelanocortin gene was converted to a premature translational stop codon. The resulting transgenic mice display no overt developmental or behavioral alterations and have a normally functioning hypothalamic-pituitary-adrenal axis. Homozygous transgenic mice with a selective deficiency of beta-endorphin exhibit normal analgesia in response to morphine, indicating the presence of functional mu-opiate receptors. However, these mice lack the opioid (naloxone reversible) analgesia induced by mild swim stress. Mutant mice also display significantly greater nonopioid analgesia in response to cold water swim stress compared with controls and display paradoxical naloxone-induced analgesia. These changes may reflect compensatory upregulation of alternative pain inhibitory mechanisms.

Codon usage affects the structure and function of the Drosophila circadian clock protein PERIOD.

PubMed

Fu, Jingjing; Murphy, Katherine A; Zhou, Mian; Li, Ying H; Lam, Vu H; Tabuloc, Christine A; Chiu, Joanna C; Liu, Yi

2016-08-01

Codon usage bias is a universal feature of all genomes, but its in vivo biological functions in animal systems are not clear. To investigate the in vivo role of codon usage in animals, we took advantage of the sensitivity and robustness of the Drosophila circadian system. By codon-optimizing parts of Drosophila period (dper), a core clock gene that encodes a critical component of the circadian oscillator, we showed that dper codon usage is important for circadian clock function. Codon optimization of dper resulted in conformational changes of the dPER protein, altered dPER phosphorylation profile and stability, and impaired dPER function in the circadian negative feedback loop, which manifests into changes in molecular rhythmicity and abnormal circadian behavioral output. This study provides an in vivo example that demonstrates the role of codon usage in determining protein structure and function in an animal system. These results suggest a universal mechanism in eukaryotes that uses a codon usage "code" within genetic codons to regulate cotranslational protein folding. © 2016 Fu et al.; Published by Cold Spring Harbor Laboratory Press.

Analyses of clinicopathological, molecular, and prognostic associations of KRAS codon 61 and codon 146 mutations in colorectal cancer: cohort study and literature review

PubMed Central

2014-01-01

Background KRAS mutations in codons 12 and 13 are established predictive biomarkers for anti-EGFR therapy in colorectal cancer. Previous studies suggest that KRAS codon 61 and 146 mutations may also predict resistance to anti-EGFR therapy in colorectal cancer. However, clinicopathological, molecular, and prognostic features of colorectal carcinoma with KRAS codon 61 or 146 mutation remain unclear. Methods We utilized a molecular pathological epidemiology database of 1267 colon and rectal cancers in the Nurse’s Health Study and the Health Professionals Follow-up Study. We examined KRAS mutations in codons 12, 13, 61 and 146 (assessed by pyrosequencing), in relation to clinicopathological features, and tumor molecular markers, including BRAF and PIK3CA mutations, CpG island methylator phenotype (CIMP), LINE-1 methylation, and microsatellite instability (MSI). Survival analyses were performed in 1067 BRAF-wild-type cancers to avoid confounding by BRAF mutation. Cox proportional hazards models were used to compute mortality hazard ratio, adjusting for potential confounders, including disease stage, PIK3CA mutation, CIMP, LINE-1 hypomethylation, and MSI. Results KRAS codon 61 mutations were detected in 19 cases (1.5%), and codon 146 mutations in 40 cases (3.2%). Overall KRAS mutation prevalence in colorectal cancers was 40% (=505/1267). Of interest, compared to KRAS-wild-type, overall, KRAS-mutated cancers more frequently exhibited cecal location (24% vs. 12% in KRAS-wild-type; P < 0.0001), CIMP-low (49% vs. 32% in KRAS-wild-type; P < 0.0001), and PIK3CA mutations (24% vs. 11% in KRAS-wild-type; P < 0.0001). These trends were evident irrespective of mutated codon, though statistical power was limited for codon 61 mutants. Neither KRAS codon 61 nor codon 146 mutation was significantly associated with clinical outcome or prognosis in univariate or multivariate analysis [colorectal cancer-specific mortality hazard ratio (HR) = 0.81, 95% confidence interval (CI) = 0.29-2.26 for codon 61 mutation; colorectal cancer-specific mortality HR = 0.86, 95% CI = 0.42-1.78 for codon 146 mutation]. Conclusions Tumors with KRAS mutations in codons 61 and 146 account for an appreciable proportion (approximately 5%) of colorectal cancers, and their clinicopathological and molecular features appear generally similar to KRAS codon 12 or 13 mutated cancers. To further assess clinical utility of KRAS codon 61 and 146 testing, large-scale trials are warranted. PMID:24885062

Codon adaptation and synonymous substitution rate in diatom plastid genes.

PubMed

Morton, Brian R; Sorhannus, Ulf; Fox, Martin

2002-07-01

Diatom plastid genes are examined with respect to codon adaptation and rates of silent substitution (Ks). It is shown that diatom genes follow the same pattern of codon usage as other plastid genes studied previously. Highly expressed diatom genes display codon adaptation, or a bias toward specific major codons, and these major codons are the same as those in red algae, green algae, and land plants. It is also found that there is a strong correlation between Ks and variation in codon adaptation across diatom genes, providing the first evidence for such a relationship in the algae. It is argued that this finding supports the notion that the correlation arises from selective constraints, not from variation in mutation rate among genes. Finally, the diatom genes are examined with respect to variation in Ks among different synonymous groups. Diatom genes with strong codon adaptation do not show the same variation in synonymous substitution rate among codon groups as the flowering plant psbA gene which, previous studies have shown, has strong codon adaptation but unusually high rates of silent change in certain synonymous groups. The lack of a similar finding in diatoms supports the suggestion that the feature is unique to the flowering plant psbA due to recent relaxations in selective pressure in that lineage.

Genetic Code Optimization for Cotranslational Protein Folding: Codon Directional Asymmetry Correlates with Antiparallel Betasheets, tRNA Synthetase Classes.

PubMed

Seligmann, Hervé; Warthi, Ganesh

2017-01-01

A new codon property, codon directional asymmetry in nucleotide content (CDA), reveals a biologically meaningful genetic code dimension: palindromic codons (first and last nucleotides identical, codon structure XZX) are symmetric (CDA = 0), codons with structures ZXX/XXZ are 5'/3' asymmetric (CDA = - 1/1; CDA = - 0.5/0.5 if Z and X are both purines or both pyrimidines, assigning negative/positive (-/+) signs is an arbitrary convention). Negative/positive CDAs associate with (a) Fujimoto's tetrahedral codon stereo-table; (b) tRNA synthetase class I/II (aminoacylate the 2'/3' hydroxyl group of the tRNA's last ribose, respectively); and (c) high/low antiparallel (not parallel) betasheet conformation parameters. Preliminary results suggest CDA-whole organism associations (body temperature, developmental stability, lifespan). Presumably, CDA impacts spatial kinetics of codon-anticodon interactions, affecting cotranslational protein folding. Some synonymous codons have opposite CDA sign (alanine, leucine, serine, and valine), putatively explaining how synonymous mutations sometimes affect protein function. Correlations between CDA and tRNA synthetase classes are weaker than between CDA and antiparallel betasheet conformation parameters. This effect is stronger for mitochondrial genetic codes, and potentially drives mitochondrial codon-amino acid reassignments. CDA reveals information ruling nucleotide-protein relations embedded in reversed (not reverse-complement) sequences (5'-ZXX-3'/5'-XXZ-3').

Development of a codon optimization strategy using the efor RED reporter gene as a test case

NASA Astrophysics Data System (ADS)

Yip, Chee-Hoo; Yarkoni, Orr; Ajioka, James; Wan, Kiew-Lian; Nathan, Sheila

2018-04-01

Synthetic biology is a platform that enables high-level synthesis of useful products such as pharmaceutically related drugs, bioplastics and green fuels from synthetic DNA constructs. Large-scale expression of these products can be achieved in an industrial compliant host such as Escherichia coli. To maximise the production of recombinant proteins in a heterologous host, the genes of interest are usually codon optimized based on the codon usage of the host. However, the bioinformatics freeware available for standard codon optimization might not be ideal in determining the best sequence for the synthesis of synthetic DNA. Synthesis of incorrect sequences can prove to be a costly error and to avoid this, a codon optimization strategy was developed based on the E. coli codon usage using the efor RED reporter gene as a test case. This strategy replaces codons encoding for serine, leucine, proline and threonine with the most frequently used codons in E. coli. Furthermore, codons encoding for valine and glycine are substituted with the second highly used codons in E. coli. Both the optimized and original efor RED genes were ligated to the pJS209 plasmid backbone using Gibson Assembly and the recombinant DNAs were transformed into E. coli E. cloni 10G strain. The fluorescence intensity per cell density of the optimized sequence was improved by 20% compared to the original sequence. Hence, the developed codon optimization strategy is proposed when designing an optimal sequence for heterologous protein production in E. coli.

Analysis of Synonymous Codon Usage Bias of Zika Virus and Its Adaption to the Hosts

PubMed Central

Wang, Hongju; Liu, Siqing; Zhang, Bo

2016-01-01

Zika virus (ZIKV) is a mosquito-borne virus (arbovirus) in the family Flaviviridae, and the symptoms caused by ZIKV infection in humans include rash, fever, arthralgia, myalgia, asthenia and conjunctivitis. Codon usage bias analysis can reveal much about the molecular evolution and host adaption of ZIKV. To gain insight into the evolutionary characteristics of ZIKV, we performed a comprehensive analysis on the codon usage pattern in 46 ZIKV strains by calculating the effective number of codons (ENc), codon adaptation index (CAI), relative synonymous codon usage (RSCU), and other indicators. The results indicate that the codon usage bias of ZIKV is relatively low. Several lines of evidence support the hypothesis that translational selection plays a role in shaping the codon usage pattern of ZIKV. The results from a correspondence analysis (CA) indicate that other factors, such as base composition, aromaticity, and hydrophobicity may also be involved in shaping the codon usage pattern of ZIKV. Additionally, the results from a comparative analysis of RSCU between ZIKV and its hosts suggest that ZIKV tends to evolve codon usage patterns that are comparable to those of its hosts. Moreover, selection pressure from Homo sapiens on the ZIKV RSCU patterns was found to be dominant compared with that from Aedes aegypti and Aedes albopictus. Taken together, both natural translational selection and mutation pressure are important for shaping the codon usage pattern of ZIKV. Our findings contribute to understanding the evolution of ZIKV and its adaption to its hosts. PMID:27893824

Control of total GFP expression by alterations to the 3′ region nucleotide sequence

PubMed Central

2013-01-01

Background Previously, we distinguished the Escherichia coli type II cytoplasmic membrane translocation pathways of Tat, Yid, and Sec for unfolded and folded soluble target proteins. The translocation of folded protein to the periplasm for soluble expression via the Tat pathway was controlled by an N-terminal hydrophilic leader sequence. In this study, we investigated the effect of the hydrophilic C-terminal end and its nucleotide sequence on total and soluble protein expression. Results The native hydrophilic C-terminal end of GFP was obtained by deleting the C-terminal peptide LeuGlu-6×His, derived from pET22b(+). The corresponding clones induced total and soluble GFP expression that was either slightly increased or dramatically reduced, apparently through reconstruction of the nucleotide sequence around the stop codon in the 3′ region. In the expression-induced clones, the hydrophilic C-terminus showed increased Tat pathway specificity for soluble expression. However, in the expression-reduced clone, after analyzing the role of the 5′ poly(A) coding sequence with a substituted synonymous codon, we proved that the longer 5′ poly(A) coding sequence interacted with the reconstructed 3′ region nucleotide sequence to create a new mRNA tertiary structure between the 5′ and 3′ regions, which resulted in reduced total GFP expression. Further, to recover the reduced expression by changing the 3′ nucleotide sequence, after replacing selected C-terminal 5′ codons and the stop codon in the ORF with synonymous codons, total GFP expression in most of the clones was recovered to the undeleted control level. The insertion of trinucleotides after the stop codon in the 3′-UTR recovered or reduced total GFP expression. RT-PCR revealed that the level of total protein expression was controlled by changes in translational or transcriptional regulation, which were induced or reduced by the substitution or insertion of 3′ region nucleotides. Conclusions We found that the hydrophilic C-terminal end of GFP increased Tat pathway specificity and that the 3′ nucleotide sequence played an important role in total protein expression through translational and transcriptional regulation. These findings may be useful for efficiently producing recombinant proteins as well as for potentially controlling the expression level of specific genes in the body for therapeutic purposes. PMID:23834827

Features of Scots pine radial growth in conditions of provenance trial.

NASA Astrophysics Data System (ADS)

Kuzmin, Sergey; Kuzmina, Nina

2013-04-01

Provenance trial of Scots pine in Boguchany forestry of Krasnoyarsk krai is conducted on two different soils - dark-grey loam forest soil and sod-podzol sandy soil. Complex of negative factors for plant growth and development appears in dry conditions of sandy soil. It could results in decrease of resistance to diseases. Sandy soils in different climatic zones have such common traits as low absorbing capacity, poorness of elemental nutrition, low microbiological activity and moisture capacity, very high water permeability. But Scots pine trees growing in such conditions could have certain advantages and perspectives of use. In the scope of climate change (global warming) the study of Scots pine growth on sandy soil become urgent because of more frequent appearance of dry seasons. Purpose of the work is revelation of radial growth features of Scots pine with different origin in dry conditions of sandy soil and assessment of external factors influence. The main feature of radial growth of majority of studied pine provenances in conditions of sandy soil is presence of significant variation of increment with distinct decline in 25-years old with loss of tree rings in a number of cases. The reason of it is complex of factors: deficit of June precipitation and next following outbreak of fungal disease. Found «frost rings» for all trees of studied clymatypes in 1992 are the consequence of temperature decline from May 21 to June 2 - from 23 down to 2 degree Celsius. Perspective climatypes with biggest radial increments and least sensitivity to fungal disease were revealed. Eniseysk and Vikhorevka (from Krasnoyarsk krai and Irkutsk oblast)provenances of pine have the biggest radial increments, the least sensitivity to Cenangium dieback and smallest increments decline. These climatypes are in the group of perspective provenances and in present time they are recommended for wide trial in the region for future use in plantation forest growing. Kandalaksha (Murmansk oblast) climatype from northern taiga with good resistance to pathogen have nonsignificant decline in radial increment during epiphytoty in comparison with local and southern climatypes. Southern Chemal provenance (Altai) after influence of Cenangium dieback has more than others losses of tree rings as the result of nonresistance to this fungal disease.

Mistletoe (Viscum album) infestation in the Scots pine stimulates drought-dependent oxidative damage in summer.

PubMed

Mutlu, Salih; Ilhan, Veli; Turkoglu, Halil Ibrahim

2016-04-01

This study sought to contribute to the understanding of the detrimental effect of the mistletoe (Viscum albumL.), a hemiparasitic plant, on the mortality of the Scots pine (Pinus sylvestrisL.). Fieldwork was conducted in the town of Kelkit (Gumushane province, Turkey) from April to October in 2013. Pine needles of similar ages were removed from the branches of mistletoe-infested and noninfested Scots pine plants, then transported to the laboratory and used as research materials. The effects of the mistletoe on the Scots pine during infestation were evaluated by determining the levels of water, electrolyte leakage (EL), malondialdehyde (MDA, being a product of lipid peroxidation) and reactive oxygen species (ROS) such as superoxide anion (O2 (-•)), hydrogen peroxide (H2O2) and hydroxyl radical ((•)OH). In addition, the activities of antioxidative enzymes such as superoxide dismutase (SOD), catalase (CAT) and peroxidase (POX) were measured in the same samples. The highest level of drought stress was found in summer (especially in August) as a result of the lowest water content in the soil and the highest average temperature occurring in these months. The drought stress induced by mistletoe infestation caused a regular decrease in water content, while it increased the levels of EL, MDA and ROS (H2O2, O2 (-•)and(•)OH). The infestation also stimulated the activities of CAT and POX, with the exception of SOD. On the other hand, in August, when the drought conditions were the harshest, the levels of EL and MDA, which are two of the most important indicator parameters for oxidative stress, as well as the levels of H2O2and(•)OH, which are two of the ROS leading to oxidative stress, reached the highest values in both infested and noninfested needles, whereas the O2 (-•)level decreased. For the same period and needles, CAT activity increased, while SOD activity decreased. Peroxidase activity, however, did not exhibit a significant change. Our findings indicate that the increased mortality of the Scots pine may result from the mistletoe-induced very severe drought stress, and that the increase in the capacity of antioxidative enzyme system does not protect the plant against oxidative stress in dry summer seasons. © The Author 2016. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

Problem-Solving Test: The Effect of Synonymous Codons on Gene Expression

ERIC Educational Resources Information Center

Szeberenyi, Jozsef

2009-01-01

Terms to be familiar with before you start to solve the test: the genetic code, codon, degenerate codons, protein synthesis, aminoacyl-tRNA, anticodon, antiparallel orientation, wobble, unambiguous codons, ribosomes, initiation, elongation and termination of translation, peptidyl transferase, translocation, degenerate oligonucleotides, green…

Codon usage regulates protein structure and function by affecting translation elongation speed in Drosophila cells.

PubMed

Zhao, Fangzhou; Yu, Chien-Hung; Liu, Yi

2017-08-21

Codon usage biases are found in all eukaryotic and prokaryotic genomes and have been proposed to regulate different aspects of translation process. Codon optimality has been shown to regulate translation elongation speed in fungal systems, but its effect on translation elongation speed in animal systems is not clear. In this study, we used a Drosophila cell-free translation system to directly compare the velocity of mRNA translation elongation. Our results demonstrate that optimal synonymous codons speed up translation elongation while non-optimal codons slow down translation. In addition, codon usage regulates ribosome movement and stalling on mRNA during translation. Finally, we show that codon usage affects protein structure and function in vitro and in Drosophila cells. Together, these results suggest that the effect of codon usage on translation elongation speed is a conserved mechanism from fungi to animals that can affect protein folding in eukaryotic organisms. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

Synonymous codon choices in the extremely GC-poor genome of Plasmodium falciparum: compositional constraints and translational selection.

PubMed

Musto, H; Romero, H; Zavala, A; Jabbari, K; Bernardi, G

1999-07-01

We have analyzed the patterns of synonymous codon preferences of the nuclear genes of Plasmodium falciparum, a unicellular parasite characterized by an extremely GC-poor genome. When all genes are considered, codon usage is strongly biased toward A and T in third codon positions, as expected, but multivariate statistical analysis detects a major trend among genes. At one end genes display codon choices determined mainly by the extreme genome composition of this parasite, and very probably their expression level is low. At the other end a few genes exhibit an increased relative usage of a particular subset of codons, many of which are C-ending. Since the majority of these few genes is putatively highly expressed, we postulate that the increased C-ending codons are translationally optimal. In conclusion, while codon usage of the majority of P. falciparum genes is determined mainly by compositional constraints, a small number of genes exhibit translational selection.

Most Used Codons per Amino Acid and per Genome in the Code of Man Compared to Other Organisms According to the Rotating Circular Genetic Code

PubMed Central

Castro-Chavez, Fernando

2011-01-01

My previous theoretical research shows that the rotating circular genetic code is a viable tool to make easier to distinguish the rules of variation applied to the amino acid exchange; it presents a precise and positional bio-mathematical balance of codons, according to the amino acids they codify. Here, I demonstrate that when using the conventional or classic circular genetic code, a clearer pattern for the human codon usage per amino acid and per genome emerges. The most used human codons per amino acid were the ones ending with the three hydrogen bond nucleotides: C for 12 amino acids and G for the remaining 8, plus one codon for arginine ending in A that was used approximately with the same frequency than the one ending in G for this same amino acid (plus *). The most used codons in man fall almost all the time at the rightmost position, clockwise, ending either in C or in G within the circular genetic code. The human codon usage per genome is compared to other organisms such as fruit flies (Drosophila melanogaster), squid (Loligo pealei), and many others. The biosemiotic codon usage of each genomic population or ‘Theme’ is equated to a ‘molecular language’. The C/U choice or difference, and the G/A difference in the third nucleotide of the most used codons per amino acid are illustrated by comparing the most used codons per genome in humans and squids. The human distribution in the third position of most used codons is a 12-8-2, C-G-A, nucleotide ending signature, while the squid distribution in the third position of most used codons was an odd, or uneven, distribution in the third position of its most used codons: 13-6-3, U-A-G, as its nucleotide ending signature. These findings may help to design computational tools to compare human genomes, to determine the exchangeability between compatible codons and amino acids, and for the early detection of incompatible changes leading to hereditary diseases. PMID:22997484

Vertebrate codon bias indicates a highly GC-rich ancestral genome.

PubMed

Nabiyouni, Maryam; Prakash, Ashwin; Fedorov, Alexei

2013-04-25

Two factors are thought to have contributed to the origin of codon usage bias in eukaryotes: 1) genome-wide mutational forces that shape overall GC-content and create context-dependent nucleotide bias, and 2) positive selection for codons that maximize efficient and accurate translation. Particularly in vertebrates, these two explanations contradict each other and cloud the origin of codon bias in the taxon. On the one hand, mutational forces fail to explain GC-richness (~60%) of third codon positions, given the GC-poor overall genomic composition among vertebrates (~40%). On the other hand, positive selection cannot easily explain strict regularities in codon preferences. Large-scale bioinformatic assessment, of nucleotide composition of coding and non-coding sequences in vertebrates and other taxa, suggests a simple possible resolution for this contradiction. Specifically, we propose that the last common vertebrate ancestor had a GC-rich genome (~65% GC). The data suggest that whole-genome mutational bias is the major driving force for generating codon bias. As the bias becomes prominent, it begins to affect translation and can result in positive selection for optimal codons. The positive selection can, in turn, significantly modulate codon preferences. Copyright © 2013 Elsevier B.V. All rights reserved.

The Relation of Codon Bias to Tissue-Specific Gene Expression in Arabidopsis thaliana

PubMed Central

Camiolo, Salvatore; Farina, Lorenzo; Porceddu, Andrea

2012-01-01

The codon composition of coding sequences plays an important role in the regulation of gene expression. Herein, we report systematic differences in the usage of synonymous codons among Arabidopsis thaliana genes that are expressed specifically in distinct tissues. Although we observed that both regionally and transcriptionally associated mutational biases were associated significantly with codon bias, they could not explain the observed differences fully. Similarly, given that transcript abundances did not account for the differences in codon usage, it is unlikely that selection for translational efficiency can account exclusively for the observed codon bias. Thus, we considered the possible evolution of codon bias as an adaptive response to the different abundances of tRNAs in different tissues. Our analysis demonstrated that in some cases, codon usage in genes that were expressed in a broad range of tissues was influenced primarily by the tissue in which the gene was expressed maximally. On the basis of this finding we propose that genes that are expressed in certain tissues might show a tissue-specific compositional signature in relation to codon usage. These findings might have implications for the design of transgenes in relation to optimizing their expression. PMID:22865738

Analysis of the synonymous codon usage bias in recently emerged enterovirus D68 strains.

PubMed

Karniychuk, Uladzimir U

2016-09-02

Understanding the codon usage pattern of a pathogen and relationship between pathogen and host's codon usage patterns has fundamental and applied interests. Enterovirus D68 (EV-D68) is an emerging pathogen with a potentially high public health significance. In the present study, the synonymous codon usage bias of 27 recently emerged, and historical EV-D68 strains was analyzed. In contrast to previously studied enteroviruses (enterovirus 71 and poliovirus), EV-D68 and human host have a high discrepancy between favored codons. Analysis of viral synonymous codon usage bias metrics, viral nucleotide/dinucleotide compositional parameters, and viral protein properties showed that mutational pressure is more involved in shaping the synonymous codon usage bias of EV-D68 than translation selection. Computation of codon adaptation indices allowed to estimate expression potential of the EV-D68 genome in several commonly used laboratory animals. This approach requires experimental validation and may provide an auxiliary tool for the rational selection of laboratory animals to model emerging viral diseases. Enterovirus D68 genome compositional and codon usage data can be useful for further pathogenesis, animal model, and vaccine design studies. Copyright © 2016 Elsevier B.V. All rights reserved.

Differences in codon bias cannot explain differences in translational power among microbes.

PubMed

Dethlefsen, Les; Schmidt, Thomas M

2005-01-06

Translational power is the cellular rate of protein synthesis normalized to the biomass invested in translational machinery. Published data suggest a previously unrecognized pattern: translational power is higher among rapidly growing microbes, and lower among slowly growing microbes. One factor known to affect translational power is biased use of synonymous codons. The correlation within an organism between expression level and degree of codon bias among genes of Escherichia coli and other bacteria capable of rapid growth is commonly attributed to selection for high translational power. Conversely, the absence of such a correlation in some slowly growing microbes has been interpreted as the absence of selection for translational power. Because codon bias caused by translational selection varies between rapidly growing and slowly growing microbes, we investigated whether observed differences in translational power among microbes could be explained entirely by differences in the degree of codon bias. Although the data are not available to estimate the effect of codon bias in other species, we developed an empirically-based mathematical model to compare the translation rate of E. coli to the translation rate of a hypothetical strain which differs from E. coli only by lacking codon bias. Our reanalysis of data from the scientific literature suggests that translational power can differ by a factor of 5 or more between E. coli and slowly growing microbial species. Using empirical codon-specific in vivo translation rates for 29 codons, and several scenarios for extrapolating from these data to estimates over all codons, we find that codon bias cannot account for more than a doubling of the translation rate in E. coli, even with unrealistic simplifying assumptions that exaggerate the effect of codon bias. With more realistic assumptions, our best estimate is that codon bias accelerates translation in E. coli by no more than 60% in comparison to microbes with very little codon bias. While codon bias confers a substantial benefit of faster translation and hence greater translational power, the magnitude of this effect is insufficient to explain observed differences in translational power among bacterial and archaeal species, particularly the differences between slowly growing and rapidly growing species. Hence, large differences in translational power suggest that the translational apparatus itself differs among microbes in ways that influence translational performance.

DsrA regulatory RNA represses both hns and rbsD mRNAs through distinct mechanisms in Escherichia coli.

PubMed

Lalaouna, David; Morissette, Audrey; Carrier, Marie-Claude; Massé, Eric

2015-10-01

The 87 nucleotide long DsrA sRNA has been mostly studied for its translational activation of the transcriptional regulator RpoS. However, it also represses hns mRNA, which encodes H-NS, a major regulator that affects expression of nearly 5% of Escherichia coli genes. A speculative model previously suggested that DsrA would block hns mRNA translation by binding simultaneously to start and stop codon regions of hns mRNA (coaxial model). Here, we show that DsrA efficiently blocked translation of hns mRNA by base-pairing immediately downstream of the start codon. In addition, DsrA induced hns mRNA degradation by actively recruiting the RNA degradosome complex. Data presented here led to a model of DsrA action on hns mRNA, which supports a canonical mechanism of sRNA-induced mRNA degradation by binding to the translation initiation region. Furthermore, using MS2-affinity purification coupled with RNA sequencing technology (MAPS), we also demonstrated that DsrA targets rbsD mRNA, involved in ribose utilization. Surprisingly, DsrA base pairs far downstream of rbsD start codon and induces rapid degradation of the transcript. Thus, our study enables us to draw an extended DsrA targetome. © 2015 John Wiley & Sons Ltd.

Transgenic rice expressing a codon-modified synthetic CP4-EPSPS confers tolerance to broad-spectrum herbicide, glyphosate.

PubMed

Chhapekar, Sushil; Raghavendrarao, Sanagala; Pavan, Gadamchetty; Ramakrishna, Chopperla; Singh, Vivek Kumar; Phanindra, Mullapudi Lakshmi Venkata; Dhandapani, Gurusamy; Sreevathsa, Rohini; Ananda Kumar, Polumetla

2015-05-01

Highly tolerant herbicide-resistant transgenic rice was developed by expressing codon-modified synthetic CP4--EPSPS. The transformants could tolerate up to 1% commercial glyphosate and has the potential to be used for DSR (direct-seeded rice). Weed infestation is one of the major biotic stress factors that is responsible for yield loss in direct-seeded rice (DSR). Herbicide-resistant rice has potential to improve the efficiency of weed management under DSR. Hence, the popular indica rice cultivar IR64, was genetically modified using Agrobacterium-mediated transformation with a codon-optimized CP4-EPSPS (5-enolpyruvylshikimate-3-phosphate synthase) gene, with N-terminal chloroplast targeting peptide from Petunia hybrida. Integration of the transgenes in the selected rice plants was confirmed by Southern hybridization and expression by Northern and herbicide tolerance assays. Transgenic plants showed EPSPS enzyme activity even at high concentrations of glyphosate, compared to untransformed control plants. T0, T1 and T2 lines were tested by herbicide bioassay and it was confirmed that the transgenic rice could tolerate up to 1% of commercial Roundup, which is five times more in dose used to kill weeds under field condition. All together, the transgenic rice plants developed in the present study could be used efficiently to overcome weed menace.

Synthetic oligonucleotide probes deduced from amino acid sequence data. Theoretical and practical considerations.

PubMed

Lathe, R

1985-05-05

Synthetic probes deduced from amino acid sequence data are widely used to detect cognate coding sequences in libraries of cloned DNA segments. The redundancy of the genetic code dictates that a choice must be made between (1) a mixture of probes reflecting all codon combinations, and (2) a single longer "optimal" probe. The second strategy is examined in detail. The frequency of sequences matching a given probe by chance alone can be determined and also the frequency of sequences closely resembling the probe and contributing to the hybridization background. Gene banks cannot be treated as random associations of the four nucleotides, and probe sequences deduced from amino acid sequence data occur more often than predicted by chance alone. Probe lengths must be increased to confer the necessary specificity. Examination of hybrids formed between unique homologous probes and their cognate targets reveals that short stretches of perfect homology occurring by chance make a significant contribution to the hybridization background. Statistical methods for improving homology are examined, taking human coding sequences as an example, and considerations of codon utilization and dinucleotide frequencies yield an overall homology of greater than 82%. Recommendations for probe design and hybridization are presented, and the choice between using multiple probes reflecting all codon possibilities and a unique optimal probe is discussed.

TTA codons in some genes prevent their expression in a class of developmental, antibiotic-negative, Streptomyces mutants.

PubMed Central

Leskiw, B K; Lawlor, E J; Fernandez-Abalos, J M; Chater, K F

1991-01-01

In Streptomyces coelicolor A3(2) and the related species Streptomyces lividans 66, aerial mycelium formation and antibiotic production are blocked by mutations in bldA, which specifies a tRNA(Leu)-like gene product which would recognize the UUA codon. Here we show that phenotypic expression of three disparate genes (carB, lacZ, and ampC) containing TTA codons depends strongly on bldA. Site-directed mutagenesis of carB, changing its two TTA codons to CTC (leucine) codons, resulted in bldA-independent expression; hence the bldA product is the principal tRNA for the UUA codon. Two other genes (hyg and aad) containing TTA codons show a medium-dependent reduction in phenotypic expression (hygromycin resistance and spectinomycin resistance, respectively) in bldA mutants. For hyg, evidence is presented that the UUA codon is probably being translated by a tRNA with an imperfectly matched anticodon, giving very low levels of gene product but relatively high resistance to hygromycin. It is proposed that TTA codons may be generally absent from genes expressed during vegetative growth and from the structural genes for differentiation and antibiotic production but present in some regulatory and resistance genes associated with the latter processes. The codon may therefore play a role in developmental regulation. Images PMID:1826053

Analysis of codon usage bias of envelope glycoprotein genes in nuclear polyhedrosis virus (NPV) and its relation to evolution.

PubMed

Zhao, Yongchao; Zheng, Hao; Xu, Anying; Yan, Donghua; Jiang, Zijian; Qi, Qi; Sun, Jingchen

2016-08-24

Analysis of codon usage bias is an extremely versatile method using in furthering understanding of the genetic and evolutionary paths of species. Codon usage bias of envelope glycoprotein genes in nuclear polyhedrosis virus (NPV) has remained largely unexplored at present. Hence, the codon usage bias of NPV envelope glycoprotein was analyzed here to reveal the genetic and evolutionary relationships between different viral species in baculovirus genus. A total of 9236 codons from 18 different species of NPV of the baculovirus genera were used to perform this analysis. Glycoprotein of NPV exhibits weaker codon usage bias. Neutrality plot analysis and correlation analysis of effective number of codons (ENC) values indicate that natural selection is the main factor influencing codon usage bias, and that the impact of mutation pressure is relatively smaller. Another cluster analysis shows that the kinship or evolutionary relationships of these viral species can be divided into two broad categories despite all of these 18 species are from the same baculovirus genus. There are many elements that can affect codon bias, such as the composition of amino acids, mutation pressure, natural selection, gene expression level, and etc. In the meantime, cluster analysis also illustrates that codon usage bias of virus envelope glycoprotein can serve as an effective means of evolutionary classification in baculovirus genus.

Efficient initiation of mammalian mRNA translation at a CUG codon.

PubMed Central

Dasso, M C; Jackson, R J

1989-01-01

Nucleotide substitutions were made at the initiation codon of an influenza virus NS cDNA clone in a vector carrying the bacteriophage T7 promoter. When capped mRNA transcripts of these constructs were translated in the rabbit reticulocyte lysate, a change in the initiation codon from...AUAAUGG...to...AUACUGG...reduced the in vitro translational efficiency by only 50-60%, and resulted in only a small increase in the yield of short products presumed to be initiated at downstream sites. Synthesis of the full-length product was initiated exclusively at the mutated codon, with negligible use either of in-frame upstream CUG or GUG codons, or of an in-frame downstream GUG codon. We conclude that CUG has the potential to function as an efficient initiation codon in mammalian systems, at least in certain contexts. Images PMID:2780285

Codon usage analysis of photolyase encoding genes of cyanobacteria inhabiting diverse habitats.

PubMed

Rajneesh; Pathak, Jainendra; Kannaujiya, Vinod K; Singh, Shailendra P; Sinha, Rajeshwar P

2017-07-01

Nucleotide and amino acid compositions were studied to determine the genomic and structural relationship of photolyase gene in freshwater, marine and hot spring cyanobacteria. Among three habitats, photolyase encoding genes from hot spring cyanobacteria were found to have highest GC content. The genomic GC content was found to influence the codon usage and amino acid variability in photolyases. The third position of codon was found to have more effect on amino acid variability in photolyases than the first and second positions of codon. The variation of amino acids Ala, Asp, Glu, Gly, His, Leu, Pro, Gln, Arg and Val in photolyases of three different habitats was found to be controlled by first position of codon (G1C1). However, second position (G2C2) of codon regulates variation of Ala, Cys, Gly, Pro, Arg, Ser, Thr and Tyr contents in photolyases. Third position (G3C3) of codon controls incorporation of amino acids such as Ala, Phe, Gly, Leu, Gln, Pro, Arg, Ser, Thr and Tyr in photolyases from three habitats. Photolyase encoding genes of hot spring cyanobacteria have 85% codons with G or C at third position, whereas marine and freshwater cyanobacteria showed 82 and 60% codons, respectively, with G or C at third position. Principal component analysis (PCA) showed that GC content has a profound effect in separating the genes along the first major axis according to their RSCU (relative synonymous codon usage) values, and neutrality analysis indicated that mutational pressure has resulted in codon bias in photolyase genes of cyanobacteria.

Stringent Nucleotide Recognition by the Ribosome at the Middle Codon Position.

PubMed

Liu, Wei; Shin, Dongwon; Ng, Martin; Sanbonmatsu, Karissa Y; Tor, Yitzhak; Cooperman, Barry S

2017-08-29

Accurate translation of the genetic code depends on mRNA:tRNA codon:anticodon base pairing. Here we exploit an emissive, isosteric adenosine surrogate that allows direct measurement of the kinetics of codon:anticodon University of California base formation during protein synthesis. Our results suggest that codon:anticodon base pairing is subject to tighter constraints at the middle position than at the 5'- and 3'-positions, and further suggest a sequential mechanism of formation of the three base pairs in the codon:anticodon helix.

Absence of opioid stress-induced analgesia in mice lacking beta-endorphin by site-directed mutagenesis.

PubMed Central

Rubinstein, M; Mogil, J S; Japón, M; Chan, E C; Allen, R G; Low, M J

1996-01-01

A physiological role for beta-endorphin in endogenous pain inhibition was investigated by targeted mutagenesis of the proopiomelanocortin gene in mouse embryonic stem cells. The tyrosine codon at position 179 of the proopiomelanocortin gene was converted to a premature translational stop codon. The resulting transgenic mice display no overt developmental or behavioral alterations and have a normally functioning hypothalamic-pituitary-adrenal axis. Homozygous transgenic mice with a selective deficiency of beta-endorphin exhibit normal analgesia in response to morphine, indicating the presence of functional mu-opiate receptors. However, these mice lack the opioid (naloxone reversible) analgesia induced by mild swim stress. Mutant mice also display significantly greater nonopioid analgesia in response to cold water swim stress compared with controls and display paradoxical naloxone-induced analgesia. These changes may reflect compensatory upregulation of alternative pain inhibitory mechanisms. Images Fig. 1 Fig. 2 PMID:8633004

Second generation codon optimized minicircle (CoMiC) for nonviral reprogramming of human adult fibroblasts.

PubMed

Diecke, Sebastian; Lisowski, Leszek; Kooreman, Nigel G; Wu, Joseph C

2014-01-01

The ability to induce pluripotency in somatic cells is one of the most important scientific achievements in the fields of stem cell research and regenerative medicine. This technique allows researchers to obtain pluripotent stem cells without the controversial use of embryos, providing a novel and powerful tool for disease modeling and drug screening approaches. However, using viruses for the delivery of reprogramming genes and transcription factors may result in integration into the host genome and cause random mutations within the target cell, thus limiting the use of these cells for downstream applications. To overcome this limitation, various non-integrating techniques, including Sendai virus, mRNA, minicircle, and plasmid-based methods, have recently been developed. Utilizing a newly developed codon optimized 4-in-1 minicircle (CoMiC), we were able to reprogram human adult fibroblasts using chemically defined media and without the need for feeder cells.

Balanced Codon Usage Optimizes Eukaryotic Translational Efficiency

PubMed Central

Qian, Wenfeng; Yang, Jian-Rong; Pearson, Nathaniel M.; Maclean, Calum; Zhang, Jianzhi

2012-01-01

Cellular efficiency in protein translation is an important fitness determinant in rapidly growing organisms. It is widely believed that synonymous codons are translated with unequal speeds and that translational efficiency is maximized by the exclusive use of rapidly translated codons. Here we estimate the in vivo translational speeds of all sense codons from the budding yeast Saccharomyces cerevisiae. Surprisingly, preferentially used codons are not translated faster than unpreferred ones. We hypothesize that this phenomenon is a result of codon usage in proportion to cognate tRNA concentrations, the optimal strategy in enhancing translational efficiency under tRNA shortage. Our predicted codon–tRNA balance is indeed observed from all model eukaryotes examined, and its impact on translational efficiency is further validated experimentally. Our study reveals a previously unsuspected mechanism by which unequal codon usage increases translational efficiency, demonstrates widespread natural selection for translational efficiency, and offers new strategies to improve synthetic biology. PMID:22479199

Tail-extension following the termination codon is critical for release of the nascent chain from membrane-bound ribosomes in a reticulocyte lysate cell-free system.

PubMed

Takahara, Michiyo; Sakaue, Haruka; Onishi, Yukiko; Yamagishi, Marifu; Kida, Yuichiro; Sakaguchi, Masao

2013-01-11

Nascent chain release from membrane-bound ribosomes by the termination codon was investigated using a cell-free translation system from rabbit supplemented with rough microsomal membrane vesicles. Chain release was extremely slow when mRNA ended with only the termination codon. Tail extension after the termination codon enhanced the release of the nascent chain. Release reached plateau levels with tail extension of 10 bases. This requirement was observed with all termination codons: TAA, TGA and TAG. Rapid release was also achieved by puromycin even in the absence of the extension. Efficient translation termination cannot be achieved in the presence of only a termination codon on the mRNA. Tail extension might be required for correct positioning of the termination codon in the ribosome and/or efficient recognition by release factors. Copyright © 2012. Published by Elsevier Inc.

A common periodic table of codons and amino acids.

PubMed

Biro, J C; Benyó, B; Sansom, C; Szlávecz, A; Fördös, G; Micsik, T; Benyó, Z

2003-06-27

A periodic table of codons has been designed where the codons are in regular locations. The table has four fields (16 places in each) one with each of the four nucleotides (A, U, G, C) in the central codon position. Thus, AAA (lysine), UUU (phenylalanine), GGG (glycine), and CCC (proline) were placed into the corners of the fields as the main codons (and amino acids) of the fields. They were connected to each other by six axes. The resulting nucleic acid periodic table showed perfect axial symmetry for codons. The corresponding amino acid table also displaced periodicity regarding the biochemical properties (charge and hydropathy) of the 20 amino acids and the position of the stop signals. The table emphasizes the importance of the central nucleotide in the codons and predicts that purines control the charge while pyrimidines determine the polarity of the amino acids. This prediction was experimentally tested.

Codon usage and amino acid usage influence genes expression level.

PubMed

Paul, Prosenjit; Malakar, Arup Kumar; Chakraborty, Supriyo

2018-02-01

Highly expressed genes in any species differ in the usage frequency of synonymous codons. The relative recurrence of an event of the favored codon pair (amino acid pairs) varies between gene and genomes due to varying gene expression and different base composition. Here we propose a new measure for predicting the gene expression level, i.e., codon plus amino bias index (CABI). Our approach is based on the relative bias of the favored codon pair inclination among the genes, illustrated by analyzing the CABI score of the Medicago truncatula genes. CABI showed strong correlation with all other widely used measures (CAI, RCBS, SCUO) for gene expression analysis. Surprisingly, CABI outperforms all other measures by showing better correlation with the wet-lab data. This emphasizes the importance of the neighboring codons of the favored codon in a synonymous group while estimating the expression level of a gene.

Large-Scale Genomic Analysis of Codon Usage in Dengue Virus and Evaluation of Its Phylogenetic Dependence

PubMed Central

Lara-Ramírez, Edgar E.; Salazar, Ma Isabel; López-López, María de Jesús; Salas-Benito, Juan Santiago; Sánchez-Varela, Alejandro

2014-01-01

The increasing number of dengue virus (DENV) genome sequences available allows identifying the contributing factors to DENV evolution. In the present study, the codon usage in serotypes 1–4 (DENV1–4) has been explored for 3047 sequenced genomes using different statistics methods. The correlation analysis of total GC content (GC) with GC content at the three nucleotide positions of codons (GC1, GC2, and GC3) as well as the effective number of codons (ENC, ENCp) versus GC3 plots revealed mutational bias and purifying selection pressures as the major forces influencing the codon usage, but with distinct pressure on specific nucleotide position in the codon. The correspondence analysis (CA) and clustering analysis on relative synonymous codon usage (RSCU) within each serotype showed similar clustering patterns to the phylogenetic analysis of nucleotide sequences for DENV1–4. These clustering patterns are strongly related to the virus geographic origin. The phylogenetic dependence analysis also suggests that stabilizing selection acts on the codon usage bias. Our analysis of a large scale reveals new feature on DENV genomic evolution. PMID:25136631

Di-codon Usage for Gene Classification

NASA Astrophysics Data System (ADS)

Nguyen, Minh N.; Ma, Jianmin; Fogel, Gary B.; Rajapakse, Jagath C.

Classification of genes into biologically related groups facilitates inference of their functions. Codon usage bias has been described previously as a potential feature for gene classification. In this paper, we demonstrate that di-codon usage can further improve classification of genes. By using both codon and di-codon features, we achieve near perfect accuracies for the classification of HLA molecules into major classes and sub-classes. The method is illustrated on 1,841 HLA sequences which are classified into two major classes, HLA-I and HLA-II. Major classes are further classified into sub-groups. A binary SVM using di-codon usage patterns achieved 99.95% accuracy in the classification of HLA genes into major HLA classes; and multi-class SVM achieved accuracy rates of 99.82% and 99.03% for sub-class classification of HLA-I and HLA-II genes, respectively. Furthermore, by combining codon and di-codon usages, the prediction accuracies reached 100%, 99.82%, and 99.84% for HLA major class classification, and for sub-class classification of HLA-I and HLA-II genes, respectively.

tRNA1Ser(G34) with the anticodon GGA can recognize not only UCC and UCU codons but also UCA and UCG codons.

PubMed

Yamada, Yuko; Matsugi, Jitsuhiro; Ishikura, Hisayuki

2003-04-15

The tRNA1Ser (anticodon VGA, V=uridin-5-oxyacetic acid) is essential for translation of the UCA codon in Escherichia coli. Here, we studied the translational abilities of serine tRNA derivatives, which have different bases from wild type at the first positions of their anticodons, using synthetic mRNAs containing the UCN (N=A, G, C, or U) codon. The tRNA1Ser(G34) having the anticodon GGA was able to read not only UCC and UCU codons but also UCA and UCG codons. This means that the formation of G-A or G-G pair allowed at the wobble position and these base pairs are noncanonical. The translational efficiency of the tRNA1Ser(G34) for UCA or UCG codon depends on the 2'-O-methylation of the C32 (Cm). The 2'-O-methylation of C32 may give rise to the space necessary for G-A or G-G base pair formation between the first position of anticodon and the third position of codon.

Comparative evolutionary genomics of Corynebacterium with special reference to codon and amino acid usage diversities.

PubMed

Pal, Shilpee; Sarkar, Indrani; Roy, Ayan; Mohapatra, Pradeep K Das; Mondal, Keshab C; Sen, Arnab

2018-02-01

The present study has been aimed to the comparative analysis of high GC composition containing Corynebacterium genomes and their evolutionary study by exploring codon and amino acid usage patterns. Phylogenetic study by MLSA approach, indel analysis and BLAST matrix differentiated Corynebacterium species in pathogenic and non-pathogenic clusters. Correspondence analysis on synonymous codon usage reveals that, gene length, optimal codon frequencies and tRNA abundance affect the gene expression of Corynebacterium. Most of the optimal codons as well as translationally optimal codons are C ending i.e. RNY (R-purine, N-any nucleotide base, and Y-pyrimidine) and reveal translational selection pressure on codon bias of Corynebacterium. Amino acid usage is affected by hydrophobicity, aromaticity, protein energy cost, etc. Highly expressed genes followed the cost minimization hypothesis and are less diverged at their synonymous positions of codons. Functional analysis of core genes shows significant difference in pathogenic and non-pathogenic Corynebacterium. The study reveals close relationship between non-pathogenic and opportunistic pathogenic Corynebaterium as well as between molecular evolution and survival niches of the organism.

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

PubMed

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

2004-01-01

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

Codon usage bias and tRNA over-expression in Buchnera aphidicola after aromatic amino acid nutritional stress on its host Acyrthosiphon pisum.

PubMed

Charles, Hubert; Calevro, Federica; Vinuelas, José; Fayard, Jean-Michel; Rahbe, Yvan

2006-01-01

Codon usage bias and relative abundances of tRNA isoacceptors were analysed in the obligate intracellular symbiotic bacterium, Buchnera aphidicola from the aphid Acyrthosiphon pisum, using a dedicated 35mer oligonucleotide microarray. Buchnera is archetypal of organisms living with minimal metabolic requirements and presents a reduced genome with high-evolutionary rate. Codonusage in Buchnera has been overcome by the high mutational bias towards AT bases. However, several lines of evidence for codon usage selection are given here. A significant correlation was found between tRNA relative abundances and codon composition of Buchnera genes. A significant codon usage bias was found for the choice of rare codons in Buchnera: C-ending codons are preferred in highly expressed genes, whereas G-ending codons are avoided. This bias is not explained by GC skew in the bacteria and might correspond to a selection for perfect matching between codon-anticodon pairs for some essential amino acids in Buchnera proteins. Nutritional stress applied to the aphid host induced a significant overexpression of most of the tRNA isoacceptors in bacteria. Although, molecular regulation of the tRNA operons in Buchnera was not investigated, a correlation between relative expression levels and organization in transcription unit was found in the genome of Buchnera.

Codon optimization of the adenoviral fiber negatively impacts structural protein expression and viral fitness

NASA Astrophysics Data System (ADS)

Villanueva, Eneko; Martí-Solano, Maria; Fillat, Cristina

2016-06-01

Codon usage adaptation of lytic viruses to their hosts is determinant for viral fitness. In this work, we analyzed the codon usage of adenoviral proteins by principal component analysis and assessed their codon adaptation to the host. We observed a general clustering of adenoviral proteins according to their function. However, there was a significant variation in the codon preference between the host-interacting fiber protein and the rest of structural late phase proteins, with a non-optimal codon usage of the fiber. To understand the impact of codon bias in the fiber, we optimized the Adenovirus-5 fiber to the codon usage of the hexon structural protein. The optimized fiber displayed increased expression in a non-viral context. However, infection with adenoviruses containing the optimized fiber resulted in decreased expression of the fiber and of wild-type structural proteins. Consequently, this led to a drastic reduction in viral release. The insertion of an exogenous optimized protein as a late gene in the adenovirus with the optimized fiber further interfered with viral fitness. These results highlight the importance of balancing codon usage in viral proteins to adequately exploit cellular resources for efficient infection and open new opportunities to regulate viral fitness for virotherapy and vaccine development.

A novel nuclear genetic code alteration in yeasts and the evolution of codon reassignment in eukaryotes

PubMed Central

Mühlhausen, Stefanie; Findeisen, Peggy; Plessmann, Uwe; Urlaub, Henning; Kollmar, Martin

2016-01-01

The genetic code is the cellular translation table for the conversion of nucleotide sequences into amino acid sequences. Changes to the meaning of sense codons would introduce errors into almost every translated message and are expected to be highly detrimental. However, reassignment of single or multiple codons in mitochondria and nuclear genomes, although extremely rare, demonstrates that the code can evolve. Several models for the mechanism of alteration of nuclear genetic codes have been proposed (including “codon capture,” “genome streamlining,” and “ambiguous intermediate” theories), but with little resolution. Here, we report a novel sense codon reassignment in Pachysolen tannophilus, a yeast related to the Pichiaceae. By generating proteomics data and using tRNA sequence comparisons, we show that Pachysolen translates CUG codons as alanine and not as the more usual leucine. The Pachysolen tRNACAG is an anticodon-mutated tRNAAla containing all major alanine tRNA recognition sites. The polyphyly of the CUG-decoding tRNAs in yeasts is best explained by a tRNA loss driven codon reassignment mechanism. Loss of the CUG-tRNA in the ancient yeast is followed by gradual decrease of respective codons and subsequent codon capture by tRNAs whose anticodon is not part of the aminoacyl-tRNA synthetase recognition region. Our hypothesis applies to all nuclear genetic code alterations and provides several testable predictions. We anticipate more codon reassignments to be uncovered in existing and upcoming genome projects. PMID:27197221

ChloroMitoCU: Codon patterns across organelle genomes for functional genomics and evolutionary applications.

PubMed

Sablok, Gaurav; Chen, Ting-Wen; Lee, Chi-Ching; Yang, Chi; Gan, Ruei-Chi; Wegrzyn, Jill L; Porta, Nicola L; Nayak, Kinshuk C; Huang, Po-Jung; Varotto, Claudio; Tang, Petrus

2017-06-01

Organelle genomes are widely thought to have arisen from reduction events involving cyanobacterial and archaeal genomes, in the case of chloroplasts, or α-proteobacterial genomes, in the case of mitochondria. Heterogeneity in base composition and codon preference has long been the subject of investigation of topics ranging from phylogenetic distortion to the design of overexpression cassettes for transgenic expression. From the overexpression point of view, it is critical to systematically analyze the codon usage patterns of the organelle genomes. In light of the importance of codon usage patterns in the development of hyper-expression organelle transgenics, we present ChloroMitoCU, the first-ever curated, web-based reference catalog of the codon usage patterns in organelle genomes. ChloroMitoCU contains the pre-compiled codon usage patterns of 328 chloroplast genomes (29,960 CDS) and 3,502 mitochondrial genomes (49,066 CDS), enabling genome-wide exploration and comparative analysis of codon usage patterns across species. ChloroMitoCU allows the phylogenetic comparison of codon usage patterns across organelle genomes, the prediction of codon usage patterns based on user-submitted transcripts or assembled organelle genes, and comparative analysis with the pre-compiled patterns across species of interest. ChloroMitoCU can increase our understanding of the biased patterns of codon usage in organelle genomes across multiple clades. ChloroMitoCU can be accessed at: http://chloromitocu.cgu.edu.tw/. © The Author 2017. Published by Oxford University Press on behalf of Kazusa DNA Research Institute.

Efficient Reassignment of a Frequent Serine Codon in Wild-Type Escherichia coli.

PubMed

Ho, Joanne M; Reynolds, Noah M; Rivera, Keith; Connolly, Morgan; Guo, Li-Tao; Ling, Jiqiang; Pappin, Darryl J; Church, George M; Söll, Dieter

2016-02-19

Expansion of the genetic code through engineering the translation machinery has greatly increased the chemical repertoire of the proteome. This has been accomplished mainly by read-through of UAG or UGA stop codons by the noncanonical aminoacyl-tRNA of choice. While stop codon read-through involves competition with the translation release factors, sense codon reassignment entails competition with a large pool of endogenous tRNAs. We used an engineered pyrrolysyl-tRNA synthetase to incorporate 3-iodo-l-phenylalanine (3-I-Phe) at a number of different serine and leucine codons in wild-type Escherichia coli. Quantitative LC-MS/MS measurements of amino acid incorporation yields carried out in a selected reaction monitoring experiment revealed that the 3-I-Phe abundance at the Ser208AGU codon in superfolder GFP was 65 ± 17%. This method also allowed quantification of other amino acids (serine, 33 ± 17%; phenylalanine, 1 ± 1%; threonine, 1 ± 1%) that compete with 3-I-Phe at both the aminoacylation and decoding steps of translation for incorporation at the same codon position. Reassignments of different serine (AGU, AGC, UCG) and leucine (CUG) codons with the matching tRNA(Pyl) anticodon variants were met with varying success, and our findings provide a guideline for the choice of sense codons to be reassigned. Our results indicate that the 3-iodo-l-phenylalanyl-tRNA synthetase (IFRS)/tRNA(Pyl) pair can efficiently outcompete the cellular machinery to reassign select sense codons in wild-type E. coli.

KRAS exon 2 codon 13 mutation is associated with a better prognosis than codon 12 mutation following lung metastasectomy in colorectal cancer

PubMed Central

Renaud, Stéphane; Guerrera, Francesco; Seitlinger, Joseph; Costardi, Lorena; Schaeffer, Mickaël; Romain, Benoit; Mossetti, Claudio; Claire-Voegeli, Anne; Filosso, Pier Luigi; Legrain, Michèle; Ruffini, Enrico; Falcoz, Pierre-Emmanuel; Oliaro, Alberto; Massard, Gilbert

2017-01-01

Introduction The utilization of molecular markers as routinely used biomarkers is steadily increasing. We aimed to evaluate the potential different prognostic values of KRAS exon 2 codons 12 and 13 after lung metastasectomy in colorectal cancer (CRC). Results KRAS codon 12 mutations were observed in 116 patients (77%), whereas codon 13 mutations were observed in 34 patients (23%). KRAS codon 13 mutations were associated with both longer time to pulmonary recurrence (TTPR) (median TTPR: 78 months (95% CI: 50.61–82.56) vs 56 months (95% CI: 68.71–127.51), P = 0.008) and improved overall survival (OS) (median OS: 82 months vs 54 months (95% CI: 48.93–59.07), P = 0.009). Multivariate analysis confirmed that codon 13 mutations were associated with better outcomes (TTPR: HR: 0.40 (95% CI: 0.17–0.93), P = 0.033); OS: HR: 0.39 (95% CI: 0.14–1.07), P = 0.07). Otherwise, no significant difference in OS (P = 0.78) or TTPR (P = 0.72) based on the type of amino-acid substitutions was observed among KRAS codon 12 mutations. Materials and Methods We retrospectively reviewed data from 525 patients who underwent a lung metastasectomy for CRC in two departments of thoracic surgery from 1998 to 2015 and focused on 150 patients that had KRAS exon 2 codon 12/13 mutations. Conclusions KRAS exon 2 codon 13 mutations, compared to codon 12 mutations, seem to be associated with better outcomes following lung metastasectomy in CRC. Prospective multicenter studies are necessary to fully understand the prognostic value of KRAS mutations in the lung metastases of CRC. PMID:27911859

Bicluster Pattern of Codon Context Usages between Flavivirus and Vector Mosquito Aedes aegypti: Relevance to Infection and Transcriptional Response of Mosquito Genes

PubMed Central

Behura, Susanta K.; Severson, David W.

2014-01-01

The mosquito Aedes aegypti is the primary vector of dengue virus (DENV) infection in most of the subtropical and tropical countries. Besides DENV, yellow fever virus (YFV) is also transmitted by A. aegypti. Susceptibility of A. aegypti to West Nile virus (WNV) has also been confirmed. Although studies have indicated correlation of codon bias between flaviviridae and their animal/insect hosts, it is not clear if codon sequences have any relation to susceptibility of A. aegypti to DENV, YFV and WNV. In the current study, usages of codon context sequences (codon pairs for neighboring amino acids) of the vector (A. aegypti) genome as well as the flaviviral genomes are investigated. We used bioinformatics methods to quantify codon context bias in a genome-wide manner of A. aegypti as well as DENV, WNV and YFV sequences. Mutual information statistics was applied to perform bicluster analysis of codon context bias between vector and flaviviral sequences. Functional relevance of the bicluster pattern was inferred from published microarray data. Our study shows that codon context bias of DENV, WNV and YFV sequences varies in a bicluster manner with that of specific sets of genes of A. aegypti. Many of these mosquito genes are known to be differentially expressed in response to flaviviral infection suggesting that codon context sequences of A. aegypti and the flaviviruses may play a role in the susceptible interaction between flaviviruses and this mosquito. The bias inusages of codon context sequences likely has a functional association with susceptibility of A. aegypti to flaviviral infection. The results from this study will allow us to conduct hypothesis driven tests to examine the role of codon contexts bias in evolution of vector-virus interactions at the molecular level. PMID:24838953

The positive regulatory function of the 5'-proximal open reading frames in GCN4 mRNA can be mimicked by heterologous, short coding sequences.

PubMed Central

Williams, N P; Mueller, P P; Hinnebusch, A G

1988-01-01

Translational control of GCN4 expression in the yeast Saccharomyces cerevisiae is mediated by multiple AUG codons present in the leader of GCN4 mRNA, each of which initiates a short open reading frame of only two or three codons. Upstream AUG codons 3 and 4 are required to repress GCN4 expression in normal growth conditions; AUG codons 1 and 2 are needed to overcome this repression in amino acid starvation conditions. We show that the regulatory function of AUG codons 1 and 2 can be qualitatively mimicked by the AUG codons of two heterologous upstream open reading frames (URFs) containing the initiation regions of the yeast genes PGK and TRP1. These AUG codons inhibit GCN4 expression when present singly in the mRNA leader; however, they stimulate GCN4 expression in derepressing conditions when inserted upstream from AUG codons 3 and 4. This finding supports the idea that AUG codons 1 and 2 function in the control mechanism as translation initiation sites and further suggests that suppression of the inhibitory effects of AUG codons 3 and 4 is a general consequence of the translation of URF 1 and 2 sequences upstream. Several observations suggest that AUG codons 3 and 4 are efficient initiation sites; however, these sequences do not act as positive regulatory elements when placed upstream from URF 1. This result suggests that efficient translation is only one of the important properties of the 5' proximal URFs in GCN4 mRNA. We propose that a second property is the ability to permit reinitiation following termination of translation and that URF 1 is optimized for this regulatory function. Images PMID:3065626

High-level tetracycline resistance mediated by efflux pumps Tet(A) and Tet(A)-1 with two start codons.

PubMed

Wang, Weixia; Guo, Qinglan; Xu, Xiaogang; Sheng, Zi-ke; Ye, Xinyu; Wang, Minggui

2014-11-01

Efflux is the most common mechanism of tetracycline resistance. Class A tetracycline efflux pumps, which often have high prevalence in Enterobacteriaceae, are encoded by tet(A) and tet(A)-1 genes. These genes have two potential start codons, GTG and ATG, located upstream of the genes. The purpose of this study was to determine the start codon(s) of the class A tetracycline resistance (tet) determinants tet(A) and tet(A)-1, and the tetracycline resistance level they mediated. Conjugation, transformation and cloning experiments were performed and the genetic environment of tet(A)-1 was analysed. The start codons in class A tet determinants were investigated by site-directed mutagenesis of ATG and GTG, the putative translation initiation codons. High-level tetracycline resistance was transferred from the clinical strain of Klebsiella pneumoniae 10-148 containing tet(A)-1 plasmid pHS27 to Escherichia coli J53 by conjugation. The transformants harbouring recombinant plasmids that carried tet(A) or tet(A)-1 exhibited tetracycline MICs of 256-512 µg ml(-1), with or without tetR(A). Once the ATG was mutated to a non-start codon, the tetracycline MICs were not changed, while the tetracycline MICs decreased from 512 to 64 µg ml(-1) following GTG mutation, and to ≤4 µg ml(-1) following mutation of both GTG and ATG. It was presumed that class A tet determinants had two start codons, which are the primary start codon GTG and secondary start codon ATG. Accordingly, two putative promoters were predicted. In conclusion, class A tet determinants can confer high-level tetracycline resistance and have two start codons. © 2014 The Authors.

Nonstructural proteins nsP3 and nsP4 of Ross River and O'Nyong-nyong viruses: sequence and comparison with those of other alphaviruses.

PubMed

Strauss, E G; Levinson, R; Rice, C M; Dalrymple, J; Strauss, J H

1988-05-01

We have sequenced the nsP3 and nsP4 region of two alphaviruses, Ross River virus and O'Nyong-nyong virus, in order to examine these viruses for the presence or absence of an opal termination codon present between nsP3 and nsP4 in many alphaviruses. We found that Ross River virus possesses an in-phase opal termination codon between nsP3 and nsP4, whereas in O'Nyong-nyong virus this termination codon is replaced by an arginine codon. Previous studies have shown that two other alphaviruses, Sindbis virus and Middelburg virus, possess an opal termination codon separating nsP3 and nsP4 [E.G. Strauss, C.M. Rice, and J.H. Strauss (1983), Proc. Natl. Acad. Sci. USA 80, 5271-5275], whereas Semliki Forest virus possesses an arginine codon in lieu of the opal codon [K. Takkinen (1986), Nucleic Acids Res. 14, 5667-5682]. Thus, of the five alphaviruses examined to date, three possess the opal codon and two do not. Production of nsP4 requires readthrough of the opal codon in those alphaviruses that possess this termination codon and the function of the termination codon may be to regulate the amount of nsP4 produced. It is an open question then as to whether alphaviruses with no termination codon use other mechanisms to regulate the activity of this gene. The nsP4s of these five alphaviruses are highly conserved, sharing 71-76% amino acid sequence similarity, and all five contain the Gly-Asp-Asp motif found in many RNA virus replicases. The nsP3s are somewhat less conserved, sharing 52-73% amino acid sequence similarity throughout most of the protein, but each possesses a nonconserved C-terminal domain of 134 to 246 amino acids of unknown function.

Non-uniqueness of factors constraint on the codon usage in Bombyx mori.

PubMed

Jia, Xian; Liu, Shuyu; Zheng, Hao; Li, Bo; Qi, Qi; Wei, Lei; Zhao, Taiyi; He, Jian; Sun, Jingchen

2015-05-06

The analysis of codon usage is a good way to understand the genetic and evolutionary characteristics of an organism. However, there are only a few reports related with the codon usage of the domesticated silkworm, Bombyx mori (B. mori). Hence, the codon usage of B. mori was analyzed here to reveal the constraint factors and it could be helpful to improve the bioreactor based on B. mori. A total of 1,097 annotated mRNA sequences from B. mori were analyzed, revealing there is only a weak codon bias. It also shows that the gene expression level is related to the GC content, and the amino acids with higher general average hydropathicity (GRAVY) and aromaticity (Aromo). And the genes on the primary axis are strongly positively correlated with the GC content, and GC3s. Meanwhile, the effective number of codons (ENc) is strongly correlated with codon adaptation index (CAI), gene length, and Aromo values. However, the ENc values are correlated with the second axis, which indicates that the codon usage in B. mori is affected by not only mutation pressure and natural selection, but also nucleotide composition and the gene expression level. It is also associated with Aromo values, and gene length. Additionally, B. mori has a greater relative discrepancy in codon preferences with Drosophila melanogaster (D. melanogaster) or Saccharomyces cerevisiae (S. cerevisiae) than with Arabidopsis thaliana (A. thaliana), Escherichia coli (E. coli), or Caenorhabditis elegans (C. elegans). The codon usage bias in B. mori is relatively weak, and many influence factors are found here, such as nucleotide composition, mutation pressure, natural selection, and expression level. Additionally, it is also associated with Aromo values, and gene length. Among them, natural selection might play a major role. Moreover, the "optimal codons" of B. mori are all encoded by G and C, which provides useful information for enhancing the gene expression in B. mori through codon optimization.

Restoration of chemosensitivity in cancer cells with MDR phenotype by deoxyribozyme, compared with ribozyme.

PubMed

Xing, Ai-Yan; Shi, Duan-bo; Liu, Wei; Chen, Xu; Sun, Yan-Lin; Wang, Xiao; Zhang, Jian-ping; Gao, Peng

2013-06-01

One of the main mechanisms for multidrug resistance (MDR) involves multidrug resistance gene 1 (MDR1) which encodes P-glycoprotein (Pgp). Pgp acts as a drug efflux pump and exports chemotherapeutic agents from cancer cells. Specific inhibition of Pgp expression by gene therapy is considered a well-respective strategy having less innate toxicities. At present, the investigation of DRz in reversal MDR is scarce. In the study, phosphorothioate DRz that targets to the translation initiation codon AUG was synthesized and transfected into breast cancer cells and leukemia cells with MDR phenotype. ASODN (antisense oligonucleotide) and ribozyme targets to the same region were also synthesized for comparison analysis. Alterations in MDR1 mRNA and Pgp were determined by RT-PCR, Northern blot, flow cytometry and Rh123 retention tests. Chemosensitivity of the treated cells was determined by MTT assay. The results showed that DRz could significantly suppress expression of MDR1 mRNA and inhibit synthesis of Pgp. The efflux activity of Pgp was inhibited accordingly. Chemosensitivity assay showed that a 21-fold reduction in drug resistance for Adriamycin and a 45-fold reduction in drug resistance for Vinblastine were found in the treated cells 36h after transfection. These data suggest that DRz targeted to the translation initiation codon AUG can reverse MDR phenotype in cancer cells and restore their chemosensitivity. Moreover, the reversal efficiency of DRz is better than that of ribozyme and ASODN targets to the same region of MDR1 mRNA. Copyright © 2013 Elsevier Inc. All rights reserved.

Evolution of Transcription Activator-Like Effectors in Xanthomonas oryzae

PubMed Central

Erkes, Annett; Reschke, Maik; Boch, Jens

2017-01-01

Abstract Transcription activator-like effectors (TALEs) are secreted by plant–pathogenic Xanthomonas bacteria into plant cells where they act as transcriptional activators and, hence, are major drivers in reprogramming the plant for the benefit of the pathogen. TALEs possess a highly repetitive DNA-binding domain of typically 34 amino acid (AA) tandem repeats, where AA 12 and 13, termed repeat variable di-residue (RVD), determine target specificity. Different Xanthomonas strains possess different repertoires of TALEs. Here, we study the evolution of TALEs from the level of RVDs determining target specificity down to the level of DNA sequence with focus on rice-pathogenic Xanthomonas oryzae pv. oryzae (Xoo) and Xanthomonas oryzae pv. oryzicola (Xoc) strains. We observe that codon pairs coding for individual RVDs are conserved to a similar degree as the flanking repeat sequence. We find strong indications that TALEs may evolve 1) by base substitutions in codon pairs coding for RVDs, 2) by recombination of N-terminal or C-terminal regions of existing TALEs, or 3) by deletion of individual TALE repeats, and we propose possible mechanisms. We find indications that the reassortment of TALE genes in clusters is mediated by an integron-like mechanism in Xoc. We finally study the effect of the presence/absence and evolutionary modifications of TALEs on transcriptional activation of putative target genes in rice, and find that even single RVD swaps may lead to considerable differences in activation. This correlation allowed a refined prediction of TALE targets, which is the crucial step to decipher their virulence activity. PMID:28637323

Functional Multi-Locus QTL Mapping of Temporal Trends in Scots Pine Wood Traits

PubMed Central

Li, Zitong; Hallingbäck, Henrik R.; Abrahamsson, Sara; Fries, Anders; Gull, Bengt Andersson; Sillanpää, Mikko J.; García-Gil, M. Rosario

2014-01-01

Quantitative trait loci (QTL) mapping of wood properties in conifer species has focused on single time point measurements or on trait means based on heterogeneous wood samples (e.g., increment cores), thus ignoring systematic within-tree trends. In this study, functional QTL mapping was performed for a set of important wood properties in increment cores from a 17-yr-old Scots pine (Pinus sylvestris L.) full-sib family with the aim of detecting wood trait QTL for general intercepts (means) and for linear slopes by increasing cambial age. Two multi-locus functional QTL analysis approaches were proposed and their performances were compared on trait datasets comprising 2 to 9 time points, 91 to 455 individual tree measurements and genotype datasets of amplified length polymorphisms (AFLP), and single nucleotide polymorphism (SNP) markers. The first method was a multilevel LASSO analysis whereby trend parameter estimation and QTL mapping were conducted consecutively; the second method was our Bayesian linear mixed model whereby trends and underlying genetic effects were estimated simultaneously. We also compared several different hypothesis testing methods under either the LASSO or the Bayesian framework to perform QTL inference. In total, five and four significant QTL were observed for the intercepts and slopes, respectively, across wood traits such as earlywood percentage, wood density, radial fiberwidth, and spiral grain angle. Four of these QTL were represented by candidate gene SNPs, thus providing promising targets for future research in QTL mapping and molecular function. Bayesian and LASSO methods both detected similar sets of QTL given datasets that comprised large numbers of individuals. PMID:25305041

Functional multi-locus QTL mapping of temporal trends in Scots pine wood traits.

PubMed

Li, Zitong; Hallingbäck, Henrik R; Abrahamsson, Sara; Fries, Anders; Gull, Bengt Andersson; Sillanpää, Mikko J; García-Gil, M Rosario

2014-10-09

Quantitative trait loci (QTL) mapping of wood properties in conifer species has focused on single time point measurements or on trait means based on heterogeneous wood samples (e.g., increment cores), thus ignoring systematic within-tree trends. In this study, functional QTL mapping was performed for a set of important wood properties in increment cores from a 17-yr-old Scots pine (Pinus sylvestris L.) full-sib family with the aim of detecting wood trait QTL for general intercepts (means) and for linear slopes by increasing cambial age. Two multi-locus functional QTL analysis approaches were proposed and their performances were compared on trait datasets comprising 2 to 9 time points, 91 to 455 individual tree measurements and genotype datasets of amplified length polymorphisms (AFLP), and single nucleotide polymorphism (SNP) markers. The first method was a multilevel LASSO analysis whereby trend parameter estimation and QTL mapping were conducted consecutively; the second method was our Bayesian linear mixed model whereby trends and underlying genetic effects were estimated simultaneously. We also compared several different hypothesis testing methods under either the LASSO or the Bayesian framework to perform QTL inference. In total, five and four significant QTL were observed for the intercepts and slopes, respectively, across wood traits such as earlywood percentage, wood density, radial fiberwidth, and spiral grain angle. Four of these QTL were represented by candidate gene SNPs, thus providing promising targets for future research in QTL mapping and molecular function. Bayesian and LASSO methods both detected similar sets of QTL given datasets that comprised large numbers of individuals. Copyright © 2014 Li et al.

Stringent Nucleotide Recognition by the Ribosome at the Middle Codon Position

PubMed Central

Liu, Wei; Shin, Dongwon; Ng, Martin; Sanbonmatsu, Karissa Y.; Tor, Yitzhak; Cooperman, Barry S.

2017-01-01

Accurate translation of the genetic code depends on mRNA:tRNA codon:anticodon base pairing. Here we exploit an emissive, isosteric adenosine surrogate that allows direct measurement of the kinetics of codon:anticodon base formation during protein synthesis. Our results suggest that codon:anticodon base pairing is subject to tighter constraints at the middle position than at the 5′- and 3′-positions, and further suggest a sequential mechanism of formation of the three base pairs in the codon:anticodon helix. PMID:28850078

Non-null full field X-ray mirror metrology using SCOTS: a reflection deflectometry approach

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

Su P.; Kaznatcheev K.; Wang, Y.

In a previous paper, the University of Arizona (UA) has developed a measurement technique called: Software Configurable Optical Test System (SCOTS) based on the principle of reflection deflectometry. In this paper, we present results of this very efficient optical metrology method applied to the metrology of X-ray mirrors. We used this technique to measure surface slope errors with precision and accuracy better than 100 nrad (rms) and {approx}200 nrad (rms), respectively, with a lateral resolution of few mm or less. We present results of the calibration of the metrology systems, discuss their accuracy and address the precision in measuring amore » spherical mirror.« less

A novel nuclear genetic code alteration in yeasts and the evolution of codon reassignment in eukaryotes.

PubMed

Mühlhausen, Stefanie; Findeisen, Peggy; Plessmann, Uwe; Urlaub, Henning; Kollmar, Martin

2016-07-01

The genetic code is the cellular translation table for the conversion of nucleotide sequences into amino acid sequences. Changes to the meaning of sense codons would introduce errors into almost every translated message and are expected to be highly detrimental. However, reassignment of single or multiple codons in mitochondria and nuclear genomes, although extremely rare, demonstrates that the code can evolve. Several models for the mechanism of alteration of nuclear genetic codes have been proposed (including "codon capture," "genome streamlining," and "ambiguous intermediate" theories), but with little resolution. Here, we report a novel sense codon reassignment in Pachysolen tannophilus, a yeast related to the Pichiaceae. By generating proteomics data and using tRNA sequence comparisons, we show that Pachysolen translates CUG codons as alanine and not as the more usual leucine. The Pachysolen tRNACAG is an anticodon-mutated tRNA(Ala) containing all major alanine tRNA recognition sites. The polyphyly of the CUG-decoding tRNAs in yeasts is best explained by a tRNA loss driven codon reassignment mechanism. Loss of the CUG-tRNA in the ancient yeast is followed by gradual decrease of respective codons and subsequent codon capture by tRNAs whose anticodon is not part of the aminoacyl-tRNA synthetase recognition region. Our hypothesis applies to all nuclear genetic code alterations and provides several testable predictions. We anticipate more codon reassignments to be uncovered in existing and upcoming genome projects. © 2016 Mühlhausen et al.; Published by Cold Spring Harbor Laboratory Press.

A Major Controversy in Codon-Anticodon Adaptation Resolved by a New Codon Usage Index

PubMed Central

Xia, Xuhua

2015-01-01

Two alternative hypotheses attribute different benefits to codon-anticodon adaptation. The first assumes that protein production is rate limited by both initiation and elongation and that codon-anticodon adaptation would result in higher elongation efficiency and more efficient and accurate protein production, especially for highly expressed genes. The second claims that protein production is rate limited only by initiation efficiency but that improved codon adaptation and, consequently, increased elongation efficiency have the benefit of increasing ribosomal availability for global translation. To test these hypotheses, a recent study engineered a synthetic library of 154 genes, all encoding the same protein but differing in degrees of codon adaptation, to quantify the effect of differential codon adaptation on protein production in Escherichia coli. The surprising conclusion that “codon bias did not correlate with gene expression” and that “translation initiation, not elongation, is rate-limiting for gene expression” contradicts the conclusion reached by many other empirical studies. In this paper, I resolve the contradiction by reanalyzing the data from the 154 sequences. I demonstrate that translation elongation accounts for about 17% of total variation in protein production and that the previous conclusion is due to the use of a codon adaptation index (CAI) that does not account for the mutation bias in characterizing codon adaptation. The effect of translation elongation becomes undetectable only when translation initiation is unrealistically slow. A new index of translation elongation ITE is formulated to facilitate studies on the efficiency and evolution of the translation machinery. PMID:25480780

Exploring synonymous codon usage preferences of disulfide-bonded and non-disulfide bonded cysteines in the E. coli genome.

PubMed

Song, Jiangning; Wang, Minglei; Burrage, Kevin

2006-07-21

High-quality data about protein structures and their gene sequences are essential to the understanding of the relationship between protein folding and protein coding sequences. Firstly we constructed the EcoPDB database, which is a high-quality database of Escherichia coli genes and their corresponding PDB structures. Based on EcoPDB, we presented a novel approach based on information theory to investigate the correlation between cysteine synonymous codon usages and local amino acids flanking cysteines, the correlation between cysteine synonymous codon usages and synonymous codon usages of local amino acids flanking cysteines, as well as the correlation between cysteine synonymous codon usages and the disulfide bonding states of cysteines in the E. coli genome. The results indicate that the nearest neighboring residues and their synonymous codons of the C-terminus have the greatest influence on the usages of the synonymous codons of cysteines and the usage of the synonymous codons has a specific correlation with the disulfide bond formation of cysteines in proteins. The correlations may result from the regulation mechanism of protein structures at gene sequence level and reflect the biological function restriction that cysteines pair to form disulfide bonds. The results may also be helpful in identifying residues that are important for synonymous codon selection of cysteines to introduce disulfide bridges in protein engineering and molecular biology. The approach presented in this paper can also be utilized as a complementary computational method and be applicable to analyse the synonymous codon usages in other model organisms.

Developmental stage related patterns of codon usage and genomic GC content: searching for evolutionary fingerprints with models of stem cell differentiation

PubMed Central

2007-01-01

Background The usage of synonymous codons shows considerable variation among mammalian genes. How and why this usage is non-random are fundamental biological questions and remain controversial. It is also important to explore whether mammalian genes that are selectively expressed at different developmental stages bear different molecular features. Results In two models of mouse stem cell differentiation, we established correlations between codon usage and the patterns of gene expression. We found that the optimal codons exhibited variation (AT- or GC-ending codons) in different cell types within the developmental hierarchy. We also found that genes that were enriched (developmental-pivotal genes) or specifically expressed (developmental-specific genes) at different developmental stages had different patterns of codon usage and local genomic GC (GCg) content. Moreover, at the same developmental stage, developmental-specific genes generally used more GC-ending codons and had higher GCg content compared with developmental-pivotal genes. Further analyses suggest that the model of translational selection might be consistent with the developmental stage-related patterns of codon usage, especially for the AT-ending optimal codons. In addition, our data show that after human-mouse divergence, the influence of selective constraints is still detectable. Conclusion Our findings suggest that developmental stage-related patterns of gene expression are correlated with codon usage (GC3) and GCg content in stem cell hierarchies. Moreover, this paper provides evidence for the influence of natural selection at synonymous sites in the mouse genome and novel clues for linking the molecular features of genes to their patterns of expression during mammalian ontogenesis. PMID:17349061

Characterization of codon usage pattern and influencing factors in Japanese encephalitis virus.

PubMed

Singh, Niraj K; Tyagi, Anuj; Kaur, Rajinder; Verma, Ramneek; Gupta, Praveen K

2016-08-02

Recently, several outbreaks of Japanese encephalitis (JE), caused by Japanese encephalitis virus (JEV), have been reported and it has become cause of concern across the world. In this study, detailed analysis of JEV codon usage pattern was performed. The relative synonymous codon usage (RSCU) values along with mean effective number of codons (ENC) value of 55.30 indicated the presence of low codon usages bias in JEV. The effect of mutational pressure on codon usage bias was confirmed by significant correlations of A3s, U3s, G3s, C3s, GC3s, ENC values, with overall nucleotide contents (A%, U%, G%, C%, and GC%). The correlation analysis of A3s, U3s, G3s, C3s, GC3s, with axis values of correspondence analysis (CoA) further confirmed the role of mutational pressure. However, the correlation analysis of Gravy values and Aroma values with A3s, U3s, G3s, C3s, and GC3s, indicated the presence of natural selection on codon usage bias in addition to mutational pressure. The natural selection was further confirmed by codon adaptation index (CAI) analysis. Additionally, relative dinucleotide frequencies, geographical distribution, and evolutionary processes also influenced the codon usage pattern to some extent. Copyright © 2016 Elsevier B.V. All rights reserved.

tRNA-mediated codon-biased translation in mycobacterial hypoxic persistence

NASA Astrophysics Data System (ADS)

Chionh, Yok Hian; McBee, Megan; Babu, I. Ramesh; Hia, Fabian; Lin, Wenwei; Zhao, Wei; Cao, Jianshu; Dziergowska, Agnieszka; Malkiewicz, Andrzej; Begley, Thomas J.; Alonso, Sylvie; Dedon, Peter C.

2016-11-01

Microbial pathogens adapt to the stress of infection by regulating transcription, translation and protein modification. We report that changes in gene expression in hypoxia-induced non-replicating persistence in mycobacteria--which models tuberculous granulomas--are partly determined by a mechanism of tRNA reprogramming and codon-biased translation. Mycobacterium bovis BCG responded to each stage of hypoxia and aerobic resuscitation by uniquely reprogramming 40 modified ribonucleosides in tRNA, which correlate with selective translation of mRNAs from families of codon-biased persistence genes. For example, early hypoxia increases wobble cmo5U in tRNAThr(UGU), which parallels translation of transcripts enriched in its cognate codon, ACG, including the DosR master regulator of hypoxic bacteriostasis. Codon re-engineering of dosR exaggerates hypoxia-induced changes in codon-biased DosR translation, with altered dosR expression revealing unanticipated effects on bacterial survival during hypoxia. These results reveal a coordinated system of tRNA modifications and translation of codon-biased transcripts that enhance expression of stress response proteins in mycobacteria.

tRNA-mediated codon-biased translation in mycobacterial hypoxic persistence

PubMed Central

Chionh, Yok Hian; McBee, Megan; Babu, I. Ramesh; Hia, Fabian; Lin, Wenwei; Zhao, Wei; Cao, Jianshu; Dziergowska, Agnieszka; Malkiewicz, Andrzej; Begley, Thomas J.; Alonso, Sylvie; Dedon, Peter C.

2016-01-01

Microbial pathogens adapt to the stress of infection by regulating transcription, translation and protein modification. We report that changes in gene expression in hypoxia-induced non-replicating persistence in mycobacteria—which models tuberculous granulomas—are partly determined by a mechanism of tRNA reprogramming and codon-biased translation. Mycobacterium bovis BCG responded to each stage of hypoxia and aerobic resuscitation by uniquely reprogramming 40 modified ribonucleosides in tRNA, which correlate with selective translation of mRNAs from families of codon-biased persistence genes. For example, early hypoxia increases wobble cmo5U in tRNAThr(UGU), which parallels translation of transcripts enriched in its cognate codon, ACG, including the DosR master regulator of hypoxic bacteriostasis. Codon re-engineering of dosR exaggerates hypoxia-induced changes in codon-biased DosR translation, with altered dosR expression revealing unanticipated effects on bacterial survival during hypoxia. These results reveal a coordinated system of tRNA modifications and translation of codon-biased transcripts that enhance expression of stress response proteins in mycobacteria. PMID:27834374

Codon optimization underpins generalist parasitism in fungi

PubMed Central

Badet, Thomas; Peyraud, Remi; Mbengue, Malick; Navaud, Olivier; Derbyshire, Mark; Oliver, Richard P; Barbacci, Adelin; Raffaele, Sylvain

2017-01-01

The range of hosts that parasites can infect is a key determinant of the emergence and spread of disease. Yet, the impact of host range variation on the evolution of parasite genomes remains unknown. Here, we show that codon optimization underlies genome adaptation in broad host range parasites. We found that the longer proteins encoded by broad host range fungi likely increase natural selection on codon optimization in these species. Accordingly, codon optimization correlates with host range across the fungal kingdom. At the species level, biased patterns of synonymous substitutions underpin increased codon optimization in a generalist but not a specialist fungal pathogen. Virulence genes were consistently enriched in highly codon-optimized genes of generalist but not specialist species. We conclude that codon optimization is related to the capacity of parasites to colonize multiple hosts. Our results link genome evolution and translational regulation to the long-term persistence of generalist parasitism. DOI: http://dx.doi.org/10.7554/eLife.22472.001 PMID:28157073

Synonymous codon changes in the oncogenes of the cottontail rabbit papillomavirus lead to increased oncogenicity and immunogenicity of the virus

PubMed Central

Cladel, Nancy M.; Budgeon, Lynn R.; Hu, Jiafen; Balogh, Karla K.; Christensen, Neil D.

2013-01-01

Papillomaviruses use rare codons with respect to the host. The reasons for this are incompletely understood but among the hypotheses is the concept that rare codons result in low protein production and this allows the virus to escape immune surveillance. We changed rare codons in the oncogenes E6 and E7 of the cottontail rabbit papillomavirus to make them more mammalian-like and tested the mutant genomes in our in vivo animal model. While the amino acid sequences of the proteins remained unchanged, the oncogenic potential of some of the altered genomes increased dramatically. In addition, increased immunogenicity, as measured by spontaneous regression, was observed as the numbers of codon changes increased. This work suggests that codon usage may modify protein production in ways that influence disease outcome and that evaluation of synonymous codons should be included in the analysis of genetic variants of infectious agents and their association with disease. PMID:23433866

Fast response of Scots pine to improved water availability reflected in tree-ring width and delta 13C.

PubMed

Eilmann, Britta; Buchmann, Nina; Siegwolf, Rolf; Saurer, Matthias; Cherubini, Paolo; Rigling, Andreas

2010-08-01

Drought-induced forest decline, like the Scots pine mortality in inner-Alpine valleys, will gain in importance as the frequency and severity of drought events are expected to increase. To understand how chronic drought affects tree growth and tree-ring delta(13)C values, we studied mature Scots pine in an irrigation experiment in an inner-Alpine valley. Tree growth and isotope analyses were carried out at the annual and seasonal scale. At the seasonal scale, maximum delta(13)C values were measured after the hottest and driest period of the year, and were associated with decreasing growth rates. Inter-annual delta(13)C values in early- and latewood showed a strong correlation with annual climatic conditions and an immediate decrease as a response to irrigation. This indicates a tight coupling between wood formation and the freshly produced assimilates for trees exposed to chronic drought. This rapid appearance of the isotopic signal is a strong indication for an immediate and direct transfer of newly synthesized assimilates for biomass production. The fast appearance and the distinct isotopic signal suggest a low availability of old stored carbohydrates. If this was a sign for C-storage depletion, an increasing mortality could be expected when stressors increase the need for carbohydrate for defence, repair or regeneration.

Comparison of the pyrolysis behavior of lignins from different tree species.

PubMed

Wang, Shurong; Wang, Kaige; Liu, Qian; Gu, Yueling; Luo, Zhongyang; Cen, Kefa; Fransson, Torsten

2009-01-01

Despite the increasing importance of biomass pyrolysis, little is known about the pyrolysis behavior of lignin--one of the main components of biomass--due to its structural complexity and the difficulty in its isolation. In the present study, we extracted lignins from Manchurian ash (Fraxinus mandschurica) and Mongolian Scots pine (Pinus sylvestris var. mongolica) using the Bjorkman procedure, which has little effect on the structure of lignin. Fourier transform infrared (FTIR) spectrometry was used to characterize the microstructure of the Bjorkman lignins, i.e., milled wood lignins (MWLs), from the different tree species. The pyrolysis characteristics of MWLs were investigated using a thermogravimetric analyzer, and the release of the main volatile and gaseous products of pyrolysis were detected by FTIR spectroscopy. During the pyrolysis process, MWLs underwent thermo-degradation over a wide temperature range. Manchurian ash MWL showed a much higher thermal degradation rate than Mongolian Scots pine MWL in the temperature range from 290-430 degrees C. High residue yields were achieved at 37 wt.% for Mongolian Scots pine MWL and 26 wt.% for Manchurian ash MWL. In order to further investigate the mechanisms of lignin pyrolysis, we also analyzed the FTIR profiles for the main pyrolysis products (CO(2), CO, methane, methanol, phenols and formaldehyde) and investigated the variation in pyrolysis products between the different MWLs.

Differences in delta13C and diameter growth among remnant Scots pine populations in Scotland.

PubMed

Brendel, Oliver; Handley, Linda; Griffiths, Howard

2002-10-01

Published data suggest that differences in wood cellulose carbon isotope composition (delta13C) and xylem ring width among natural populations of Scots pine in Scotland (Pinus sylvestris L.) are attributable to the persistence of palaeotypes of various post-glacial migratory origins. We assessed differences in wood cellulose delta13C and ring width among Scottish Scots pine populations grown in a clone bank and in natural stands at various locations in northern and central Scotland. Ring width and wood cellulose delta13C varied significantly among natural stands. Potential water deficit was positively correlated with wood cellulose delta13C and xylem ring width in the natural stands. Neither wood cellulose delta13C nor xylem ring width of clone bank trees correlated with any climate variables at the sites from which the trees originated, indicating little adaptation to climate for these traits. Xylem ring width showed a site x population interaction for the growth sites (i.e., natural stands versus clone bank), but wood cellulose delta13C did not. These results suggest that climate variation in Scotland has not resulted in significant genetic variation in wood cellulose delta13C or xylem ring width in post-glacial populations.

Drought alters timing, quantity, and quality of wood formation in Scots pine.

PubMed

Eilmann, Britta; Zweifel, Roman; Buchmann, Nina; Graf Pannatier, Elisabeth; Rigling, Andreas

2011-05-01

Drought has been frequently discussed as a trigger for forest decline. Today, large-scale Scots pine decline is observed in many dry inner-Alpine valleys, with drought discussed as the main causative factor. This study aimed to analyse the impact of drought on wood formation and wood structure. To study tree growth under contrasting water supply, an irrigation experiment was installed in a mature Scots pine (Pinus sylvestris L.) forest at a xeric site in a dry inner-Alpine valley. Inter- and intra-annual radial increments as well as intra-annual variations in wood structure of pine trees were studied. It was found that non-irrigated trees had a noticeably shorter period of wood formation and showed a significantly lower increment. The water conduction cells were significantly enlarged and had significantly thinner cell walls compared with irrigated trees. It is concluded that pine trees under drought stress build a more effective water-conducting system (larger tracheids) at the cost of a probably higher vulnerability to cavitation (larger tracheids with thinner cell walls) but without losing their capability to recover. The significant shortening of the growth period in control trees indicated that the period where wood formation actually takes place can be much shorter under drought than the 'potential' period, meaning the phenological growth period.

Climate influences the leaf area/sapwood area ratio in Scots pine.

PubMed

Mencuccini, M; Grace, J

1995-01-01

We tested the hypothesis that the leaf area/sapwood area ratio in Scots pine (Pinus sylvestris L.) is influenced by site differences in water vapor pressure deficit of the air (D). Two stands of the same provenance were selected, one in western Scotland and one in eastern England, so that effects resulting from age, genetic variability, density and fertility were minimized. Compared with the Scots pine trees at the cooler and wetter site in Scotland, the trees at the warmer and drier site in England produced less leaf area per unit of conducting sapwood area both at a stem height of 1.3 m and at the base of the live crown, whereas stem permeability was similar at both sites. Also, trees at the drier site had less leaf area per unit branch cross-sectional area at the branch base than trees at the wetter site. For each site, the average values for leaf area, sapwood area and permeability were used, together with values of transpiration rates at different D, to calculate average stem water potential gradients. Changes in the leaf area/sapwood area ratio acted to maintain a similar water potential gradient in the stems of trees at both sites despite climatic differences between the sites.

Estimating the onset of cambial activity in Scots pine in northern Finland by means of the heat-sum approach.

PubMed

Seo, Jeong-Wook; Eckstein, Dieter; Jalkanen, Risto; Rickebusch, Sophie; Schmitt, Uwe

2008-01-01

We estimated the date of onset (Date(est)) of cambial activity by the pinning method in Scots pine (Pinus sylvestris L.) trees at Vanttauskoski (Site 1) and Laanila (Site 2) near the latitudinal limit of Scots pine in northern Finland. In each year and at each site, observations were made on a different set of five trees. The estimated dates of onset of cambial activity were compared with the corresponding heat sums, calculated in degree-days according to two models. Within years, Date(est) varied among trees by up to 15 days at Site 1 and up to 13 days at Site 2. Among years, mean Date(est) varied by 15.3 days at Site 1 and 12.0 days at Site 2. The overall mean Date(est) differed between sites by 6 days (June 5 at Site 1 and June 11 at Site 2). Among all trees in all years, the mean number of degree days (d.d.) calculated from mean daily temperature above a threshold of 5 degrees C before Date(est) ranged from 68.7 to 135 d.d. at Site 1 and from 37.4 to 154.7 d.d. at Site 2. Among years, the mean heat sum before Date(est )ranged from 94 to 112.5 d.d. at Site 1 and from 61.4 to 136 d.d. at Site 2. Variation among years in heat sum before Date(est) at Site 2 was highly significant, indicating that one or more factors other than, or in addition to, heat sum determines the onset of cambial activity in Scots pine. Similar results were obtained when heat sum was computed from the area between the sine wave generated by daily maximum and minimum temperature and the threshold temperature.

Above- and belowground fluxes of CH4 from boreal shrubs and Scots pine

NASA Astrophysics Data System (ADS)

Halmeenmäki, Elisa; Heinonsalo, Jussi; Santalahti, Minna; Putkinen, Anuliina; Fritze, Hannu; Pihlatie, Mari

2016-04-01

Boreal upland forests are considered as an important sink for the greenhouse gas methane (CH4) due to CH4 oxidizing microbes in the soil. However, recent evidence suggests that vegetation can act as a significant source of CH4. Also, preliminary measurements indicate occasional emissions of CH4 above the tree canopies of a boreal forest. Nevertheless, the sources and the mechanisms of the observed CH4 emissions are still mostly unknown. Furthermore, the majority of CH4 flux studies have been conducted with the soil chamber method, thus not considering the role of the vegetation itself. We conducted a laboratory experiment to study separately the above- and belowground CH4 fluxes of bilberry (Vaccinium myrtillus), lingonberry (Vaccinium vitis-idaea), heather (Calluna vulgaris), and Scots pine (Pinus sylvestris), which were grown in microcosms. The above- and belowground fluxes of the plants were measured separately, and these fluxes were compared to fluxes of microcosms containing only humus soil. In addition to the flux measurements, we analysed the CH4 producing archaea (methanogens) and the CH4 consuming bacteria (methanotrophs) with the qPCR method to discover whether these microbes contribute to the CH4 exchange from the plant material and the soil. The results of the flux measurements indicate that the humus soil with roots of lingonberry, heather, and Scots pine consume CH4 compared to bare humus soil. Simultaneously, the shoots of heather and Scots pine emit small amounts of CH4. We did not find detectable amounts of methanogens from any of the samples, suggesting the produced CH4 could be of non-microbial origin, or produced by very small population of methanogens. Based on the first preliminary results, methanotrophs were present in all the studied plant species, and especially in high amounts in the rooted soils, thus implying that the methanotrophs could be responsible of the CH4 uptake in the root-soil systems.

Modeling of bud break of Scots pine in northern Finland in 1908–2014

PubMed Central

Salminen, Hannu; Jalkanen, Risto

2015-01-01

Bud break and height-growth of Scots pine (Pinus sylvestris L.) in the northern boreal zone in Lapland, Finland, was followed through the entire growing seasons in the periods 2001–2003 and 2008–2010 in sapling stands in two different locations in northern Finland set some 250 km apart along a latitudinal transect. Field measurements continued at the southern site also in 2011–2013. Air temperature was recorded hourly at the sites. A simple optimization algorithm (GA) was used to adjust parameters of the models predicting the timing of bud break of Scots pine in order to minimize the difference between observed and predicted dates. The models giving the best performance and century-long daily temperatures were used to reconstruct bud-break time series. The temperature observations were recorded for the period 1908–2014 in Sodankylä, which is located in-between the sapling stands in the north–south direction and for the period 1877–2014 in Karasjok, which is in Norway about 145 km north–west from the northernmost stand of this study. On average buds began to extend in the beginning of May in the southernmost stand and in mid-May in the northernmost stands, and the variation between years was in the range of 3 weeks. A simple day-length-triggered (fixed date) model predicted most accurately the date of bud break; root mean square error (RMSE) was 2 and 4 days in the northern and southern site, respectively. The reconstructed bud-break series indicated that based on temperature observations from Sodankylä, growth onset of Scots pine has clearly advanced since the 1960s, though it currently matches that of the early 1920s and early 1950s. The temperature record from Karasjok indicated a similar variation, though there was a weak linear trend advancing bud break by about 3–4 days over a 100-year period. PMID:25798141

Levels of selected trace elements in Scots pine (Pinus sylvestris L.), silver birch (Betula pendula L.), and Norway maple (Acer platanoides L.) in an urbanized environment.

PubMed

Kosiorek, Milena; Modrzewska, Beata; Wyszkowski, Mirosław

2016-10-01

The aim of the study was to determine the concentrations of selected trace elements in needles and bark of Scots pine (Pinus sylvestris L.), leaves and bark of silver birch (Betula pendula L.), and Norway maple (Acer platanoides L.), as well as in the soil in which the trees grew, depending on their localization and hence the distribution of local pollution sources. The content of trace elements in needles of Scots pine, leaves of silver birch, and Norway maple and in bark of these trees depended on the location, tree species, and analyzed organ. The content of Fe, Mn, and Zn in needles, leaves, and bark of the examined tree species was significantly higher than that of the other elements. The highest average content of Fe and Mn was detected in leaves of Norway maple whereas the highest average content of Zn was found in silver birch leaves. The impact of such locations as the center of Olsztyn or roadside along Road 51 on the content of individual elements tended to be more pronounced than the influence of the other locations. The influence of the sampling sites on the content of trace elements in tree bark was less regular than the analogous effect in needles and leaves. Moreover, the relevant dependences were slightly different for Scots pine than for the other two tree species. The concentrations of heavy metals determined in the soil samples did not exceed the threshold values set in the Regulation of the Minister for the Environment, although the soil along Road 51 and in the center of Olsztyn typically had the highest content of these elements. There were also significant correlations between the content of some trace elements in soil and their accumulation in needles, leaves, and bark of trees.

TIP: protein backtranslation aided by genetic algorithms.

PubMed

Moreira, Andrés; Maass, Alejandro

2004-09-01

Several applications require the backtranslation of a protein sequence into a nucleic acid sequence. The degeneracy of the genetic code makes this process ambiguous; moreover, not every translation is equally viable. The usual answer is to mimic the codon usage of the target species; however, this does not capture all the relevant features of the 'genomic styles' from different taxa. The program TIP ' Traducción Inversa de Proteínas') applies genetic algorithms to improve the backtranslation, by minimizing the difference of some coding statistics with respect to their average value in the target. http://www.cmm.uchile.cl/genoma/tip/

Molecular analysis of beta-globin gene mutations among Thai beta-thalassemia children: results from a single center study

PubMed Central

Boonyawat, Boonchai; Monsereenusorn, Chalinee; Traivaree, Chanchai

2014-01-01

Background Beta-thalassemia is one of the most common genetic disorders in Thailand. Clinical phenotype ranges from silent carrier to clinically manifested conditions including severe beta-thalassemia major and mild beta-thalassemia intermedia. Objective This study aimed to characterize the spectrum of beta-globin gene mutations in pediatric patients who were followed-up in Phramongkutklao Hospital. Patients and methods Eighty unrelated beta-thalassemia patients were enrolled in this study including 57 with beta-thalassemia/hemoglobin E, eight with homozygous beta-thalassemia, and 15 with heterozygous beta-thalassemia. Mutation analysis was performed by multiplex amplification refractory mutation system (M-ARMS), direct DNA sequencing of beta-globin gene, and gap polymerase chain reaction for 3.4 kb deletion detection, respectively. Results A total of 13 different beta-thalassemia mutations were identified among 88 alleles. The most common mutation was codon 41/42 (-TCTT) (37.5%), followed by codon 17 (A>T) (26.1%), IVS-I-5 (G>C) (8%), IVS-II-654 (C>T) (6.8%), IVS-I-1 (G>T) (4.5%), and codon 71/72 (+A) (2.3%), and all these six common mutations (85.2%) were detected by M-ARMS. Six uncommon mutations (10.2%) were identified by DNA sequencing including 4.5% for codon 35 (C>A) and 1.1% initiation codon mutation (ATG>AGG), codon 15 (G>A), codon 19 (A>G), codon 27/28 (+C), and codon 123/124/125 (-ACCCCACC), respectively. The 3.4 kb deletion was detected at 4.5%. The most common genotype of beta-thalassemia major patients was codon 41/42 (-TCTT)/codon 26 (G>A) or betaE accounting for 40%. Conclusion All of the beta-thalassemia alleles have been characterized by a combination of techniques including M-ARMS, DNA sequencing, and gap polymerase chain reaction for 3.4 kb deletion detection. Thirteen mutations account for 100% of the beta-thalassemia genes among the pediatric patients in our study. PMID:25525381

Evolutionary characterization of Tembusu virus infection through identification of codon usage patterns.

PubMed

Zhou, Hao; Yan, Bing; Chen, Shun; Wang, Mingshu; Jia, Renyong; Cheng, Anchun

2015-10-01

Tembusu virus (TMUV) is a single-stranded, positive-sense RNA virus. As reported, TMUV infection has resulted in significant poultry losses, and the virus may also pose a threat to public health. To characterize TMUV evolutionarily and to understand the factors accounting for codon usage properties, we performed, for the first time, a comprehensive analysis of codon usage bias for the genomes of 60 TMUV strains. The most recently published TMUV strains were found to be widely distributed in coastal cities of southeastern China. Codon preference among TMUV genomes exhibits a low bias (effective number of codons (ENC)=53.287) and is maintained at a stable level. ENC-GC3 plots and the high correlation between composition constraints and principal component factor analysis of codon usage demonstrated that mutation pressure dominates over natural selection pressure in shaping the TMUV coding sequence composition. The high correlation between the major components of the codon usage pattern and hydrophobicity (Gravy) or aromaticity (Aromo) was obvious, indicating that properties of viral proteins also account for the observed variation in TMUV codon usage. Principal component analysis (PCA) showed that CQW1 isolated from Chongqing may have evolved from GX2013H or GX2013G isolated from Guangxi, thus indicating that TMUV likely disseminated from southeastern China to the mainland. Moreover, the preferred codons encoding eight amino acids were consistent with the optimal codons for human cells, indicating that TMUV may pose a threat to public health due to possible cross-species transmission (birds to birds or birds to humans). The results of this study not only have theoretical value for uncovering the characteristics of synonymous codon usage patterns in TMUV genomes but also have significant meaning with regard to the molecular evolutionary tendencies of TMUV. Copyright © 2015 Elsevier B.V. All rights reserved.

Preferences of AAA/AAG codon recognition by modified nucleosides, τm5s2U34 and t6A37 present in tRNALys.

PubMed

Sonawane, Kailas D; Kamble, Asmita S; Fandilolu, Prayagraj M

2017-12-27

Deficiency of 5-taurinomethyl-2-thiouridine, τm 5 s 2 U at the 34th 'wobble' position in tRNA Lys causes MERRF (Myoclonic Epilepsy with Ragged Red Fibers), a neuromuscular disease. This modified nucleoside of mt tRNA Lys , recognizes AAA/AAG codons during protein biosynthesis process. Its preference to identify cognate codons has not been studied at the atomic level. Hence, multiple MD simulations of various molecular models of anticodon stem loop (ASL) of mt tRNA Lys in presence and absence of τm 5 s 2 U 34 and N 6 -threonylcarbamoyl adenosine (t 6 A 37 ) along with AAA and AAG codons have been accomplished. Additional four MD simulations of multiple ASL mt tRNA Lys models in the context of ribosomal A-site residues have also been performed to investigate the role of A-site in recognition of AAA/AAG codons. MD simulation results show that, ASL models in presence of τm 5 s 2 U 34 and t 6 A 37 with codons AAA/AAG are more stable than the ASL lacking these modified bases. MD trajectories suggest that τm 5 s 2 U recognizes the codons initially by 'wobble' hydrogen bonding interactions, and then tRNA Lys might leave the explicit codon by a novel 'single' hydrogen bonding interaction in order to run the protein biosynthesis process smoothly. We propose this model as the 'Foot-Step Model' for codon recognition, in which the single hydrogen bond plays a crucial role. MD simulation results suggest that, tRNA Lys with τm 5 s 2 U and t 6 A recognizes AAA codon more preferably than AAG. Thus, these results reveal the consequences of τm 5 s 2 U and t 6 A in recognition of AAA/AAG codons in mitochondrial disease, MERRF.

Numeral series hidden in the distribution of atomic mass of amino acids to codon domains in the genetic code.

PubMed

Wohlin, Åsa

2015-03-21

The distribution of codons in the nearly universal genetic code is a long discussed issue. At the atomic level, the numeral series 2x(2) (x=5-0) lies behind electron shells and orbitals. Numeral series appear in formulas for spectral lines of hydrogen. The question here was if some similar scheme could be found in the genetic code. A table of 24 codons was constructed (synonyms counted as one) for 20 amino acids, four of which have two different codons. An atomic mass analysis was performed, built on common isotopes. It was found that a numeral series 5 to 0 with exponent 2/3 times 10(2) revealed detailed congruency with codon-grouped amino acid side-chains, simultaneously with the division on atom kinds, further with main 3rd base groups, backbone chains and with codon-grouped amino acids in relation to their origin from glycolysis or the citrate cycle. Hence, it is proposed that this series in a dynamic way may have guided the selection of amino acids into codon domains. Series with simpler exponents also showed noteworthy correlations with the atomic mass distribution on main codon domains; especially the 2x(2)-series times a factor 16 appeared as a conceivable underlying level, both for the atomic mass and charge distribution. Furthermore, it was found that atomic mass transformations between numeral systems, possibly interpretable as dimension degree steps, connected the atomic mass of codon bases with codon-grouped amino acids and with the exponent 2/3-series in several astonishing ways. Thus, it is suggested that they may be part of a deeper reference system. Copyright © 2015 The Author. Published by Elsevier Ltd.. All rights reserved.

The Enterococcus faecalis EbpA Pilus Protein: Attenuation of Expression, Biofilm Formation, and Adherence to Fibrinogen Start with the Rare Initiation Codon ATT

PubMed Central

Montealegre, Maria Camila; La Rosa, Sabina Leanti; Roh, Jung Hyeob; Harvey, Barrett R.

2015-01-01

ABSTRACT The endocarditis and biofilm-associated pili (Ebp) are important in Enterococcus faecalis pathogenesis, and the pilus tip, EbpA, has been shown to play a major role in pilus biogenesis, biofilm formation, and experimental infections. Based on in silico analyses, we previously predicted that ATT is the EbpA translational start codon, not the ATG codon, 120 bp downstream of ATT, which is annotated as the translational start. ATT is rarely used to initiate protein synthesis, leading to our hypothesis that this codon participates in translational regulation of Ebp production. To investigate this possibility, site-directed mutagenesis was used to introduce consecutive stop codons in place of two lysines at positions 5 and 6 from the ATT, to replace the ATT codon in situ with ATG, and then to revert this ATG to ATT; translational fusions of ebpA to lacZ were also constructed to investigate the effect of these start codons on translation. Our results showed that the annotated ATG does not start translation of EbpA, implicating ATT as the start codon; moreover, the presence of ATT, compared to the engineered ATG, resulted in significantly decreased EbpA surface display, attenuated biofilm, and reduced adherence to fibrinogen. Corroborating these findings, the translational fusion with the native ATT as the initiation codon showed significantly decreased expression of β-galactosidase compared to the construct with ATG in place of ATT. Thus, these results demonstrate that the rare initiation codon of EbpA negatively regulates EbpA surface display and negatively affects Ebp-associated functions, including biofilm and adherence to fibrinogen. PMID:26015496

Compositional pressure and translational selection determine codon usage in the extremely GC-poor unicellular eukaryote Entamoeba histolytica.

PubMed

Romero, H; Zavala, A; Musto, H

2000-01-25

It is widely accepted that the compositional pressure is the only factor shaping codon usage in unicellular species displaying extremely biased genomic compositions. This seems to be the case in the prokaryotes Mycoplasma capricolum, Rickettsia prowasekii and Borrelia burgdorferi (GC-poor), and in Micrococcus luteus (GC-rich). However, in the GC-poor unicellular eukaryotes Dictyostelium discoideum and Plasmodium falciparum, there is evidence that selection, acting at the level of translation, influences codon choices. This is a twofold intriguing finding, since (1) the genomic GC levels of the above mentioned eukaryotes are lower than the GC% of any studied bacteria, and (2) bacteria usually have larger effective population sizes than eukaryotes, and hence natural selection is expected to overcome more efficiently the randomizing effects of genetic drift among prokaryotes than among eukaryotes. In order to gain a new insight about this problem, we analysed the patterns of codon preferences of the nuclear genes of Entamoeba histolytica, a unicellular eukaryote characterised by an extremely AT-rich genome (GC = 25%). The overall codon usage is strongly biased towards A and T in the third codon positions, and among the presumed highly expressed sequences, there is an increased relative usage of a subset of codons, many of which are C-ending. Since an increase in C in third codon positions is 'against' the compositional bias, we conclude that codon usage in E. histolytica, as happens in D. discoideum and P. falciparum, is the result of an equilibrium between compositional pressure and selection. These findings raise the question of why strongly compositionally biased eukaryotic cells may be more sensitive to the (presumed) slight differences among synonymous codons than compositionally biased bacteria.

Intestinal cell targeting of a stable recombinant Cu-Zn SOD from Cucumis melo fused to a gliadin peptide.

PubMed

Intes, Laurent; Bahut, Muriel; Nicole, Pascal; Couvineau, Alain; Guette, Catherine; Calenda, Alphonse

2012-05-31

The mRNA encoding full length chloroplastic Cu-Zn SOD (superoxide dismutase) of Cucumis melo (Cantaloupe melon) was cloned. This sequence was then used to generate a mature recombinant SOD by deleting the first 64 codons expected to encode a chloroplastic peptide signal. A second hybrid SOD was created by inserting ten codons to encode a gliadin peptide at the N-terminal end of the mature SOD. Taking account of codon bias, both recombinant proteins were successfully expressed and produced in Escherichia coli. Both recombinant SODs display an enzymatic activity of ~5000U mg(-1) and were shown to be stable for at least 4h at 37°C in biological fluids mimicking the conditions of intestinal transit. These recombinant proteins were capable in vitro, albeit at different levels, of reducing ROS-induced-apoptosis of human epithelial cells. They also stimulated production and release in a time-dependent manner of an autologous SOD activity from cells located into jejunum biopsies. Nevertheless, the fused gliadin peptide enable the recombinant Cu-Zn SOD to maintain a sufficiently sustained interaction with the intestinal cells membrane in vivo rather than being eliminated with the flow. According to these observations, the new hybrid Cu-Zn SOD should show promise in applications for managing inflammatory bowel diseases. Copyright © 2012 Elsevier B.V. All rights reserved.

Species Based Synonymous Codon Usage in Fusion Protein Gene of Newcastle Disease Virus

PubMed Central

Kumar, Chandra Shekhar; Kumar, Sachin

2014-01-01

Newcastle disease is highly pathogenic to poultry and many other avian species. However, the Newcastle disease virus (NDV) has also been reported from many non-avian species. The NDV fusion protein (F) is a major determinant of its pathogenicity and virulence. The functionalities of F gene have been explored for the development of vaccine and diagnostics against NDV. Although the F protein is well studied but the codon usage and its nucleotide composition from NDV isolated from different species have not yet been explored. In present study, we have analyzed the factors responsible for the determination of codon usage in NDV isolated from four major avian host species. The F gene of NDV is analyzed for its base composition and its correlation with the bias in codon usage. Our result showed that random mutational pressure is responsible for codon usage bias in F protein of NDV isolates. Aromaticity, GC3s, and aliphatic index were not found responsible for species based synonymous codon usage bias in F gene of NDV. Moreover, the low amount of codon usage bias and expression level was further confirmed by a low CAI value. The phylogenetic analysis of isolates was found in corroboration with the relatedness of species based on codon usage bias. The relationship between the host species and the NDV isolates from the host does not represent a significant correlation in our study. The present study provides a basic understanding of the mechanism involved in codon usage among species. PMID:25479071

An integrated, structure- and energy-based view of the genetic code.

PubMed

Grosjean, Henri; Westhof, Eric

2016-09-30

The principles of mRNA decoding are conserved among all extant life forms. We present an integrative view of all the interaction networks between mRNA, tRNA and rRNA: the intrinsic stability of codon-anticodon duplex, the conformation of the anticodon hairpin, the presence of modified nucleotides, the occurrence of non-Watson-Crick pairs in the codon-anticodon helix and the interactions with bases of rRNA at the A-site decoding site. We derive a more information-rich, alternative representation of the genetic code, that is circular with an unsymmetrical distribution of codons leading to a clear segregation between GC-rich 4-codon boxes and AU-rich 2:2-codon and 3:1-codon boxes. All tRNA sequence variations can be visualized, within an internal structural and energy framework, for each organism, and each anticodon of the sense codons. The multiplicity and complexity of nucleotide modifications at positions 34 and 37 of the anticodon loop segregate meaningfully, and correlate well with the necessity to stabilize AU-rich codon-anticodon pairs and to avoid miscoding in split codon boxes. The evolution and expansion of the genetic code is viewed as being originally based on GC content with progressive introduction of A/U together with tRNA modifications. The representation we present should help the engineering of the genetic code to include non-natural amino acids. © The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.

Codon usage bias and tRNA over-expression in Buchnera aphidicola after aromatic amino acid nutritional stress on its host Acyrthosiphon pisum

PubMed Central

Charles, Hubert; Calevro, Federica; Vinuelas, José; Fayard, Jean-Michel; Rahbe, Yvan

2006-01-01

Codon usage bias and relative abundances of tRNA isoacceptors were analysed in the obligate intracellular symbiotic bacterium, Buchnera aphidicola from the aphid Acyrthosiphon pisum, using a dedicated 35mer oligonucleotide microarray. Buchnera is archetypal of organisms living with minimal metabolic requirements and presents a reduced genome with high-evolutionary rate. Codonusage in Buchnera has been overcome by the high mutational bias towards AT bases. However, several lines of evidence for codon usage selection are given here. A significant correlation was found between tRNA relative abundances and codon composition of Buchnera genes. A significant codon usage bias was found for the choice of rare codons in Buchnera: C-ending codons are preferred in highly expressed genes, whereas G-ending codons are avoided. This bias is not explained by GC skew in the bacteria and might correspond to a selection for perfect matching between codon–anticodon pairs for some essential amino acids in Buchnera proteins. Nutritional stress applied to the aphid host induced a significant overexpression of most of the tRNA isoacceptors in bacteria. Although, molecular regulation of the tRNA operons in Buchnera was not investigated, a correlation between relative expression levels and organization in transcription unit was found in the genome of Buchnera. PMID:16963497

Three stages during the evolution of the genetic code. [Abstract only

NASA Technical Reports Server (NTRS)

Baumann, U.; Oro, J.

1994-01-01

A diversification of the genetic code based on the number of codons available for the proteinous amino acids is established. Three groups of amino acids during evolution of the code are distinguished. On the basis of their chemical complexity and a small codon number those amino acids emerging later in a translation process are derived. Both criteria indicate that His, Phe, Tyr, Cys and either Lys or Asn were introduced in the second stage, whereas the number of codons alone gives evidence that Trp and Met were introduced in the third stage. The amino acids of stage one use purines rich codons, thus purines have been retained in their third codon position. All the amino acids introduced in the second stage, in contrast, use pyrimidines in this codon position. A low abundance of pyrimidines during early translation is derived. This assumption is supported by experiments on non enzymatic replication and interactions of DNA hairpin loops with a complementary strand. A back extrapolation concludes a high purine content of the first nucleic acids which gradually decreased during their evolution. Amino acids independently available form prebiotic synthesis were thus correlated to purine rich codons. Conclusions on prebiotic replication are discussed also in the light of recent codon usage data.

Relative codon adaptation: a generic codon bias index for prediction of gene expression.

PubMed

Fox, Jesse M; Erill, Ivan

2010-06-01

The development of codon bias indices (CBIs) remains an active field of research due to their myriad applications in computational biology. Recently, the relative codon usage bias (RCBS) was introduced as a novel CBI able to estimate codon bias without using a reference set. The results of this new index when applied to Escherichia coli and Saccharomyces cerevisiae led the authors of the original publications to conclude that natural selection favours higher expression and enhanced codon usage optimization in short genes. Here, we show that this conclusion was flawed and based on the systematic oversight of an intrinsic bias for short sequences in the RCBS index and of biases in the small data sets used for validation in E. coli. Furthermore, we reveal that how the RCBS can be corrected to produce useful results and how its underlying principle, which we here term relative codon adaptation (RCA), can be made into a powerful reference-set-based index that directly takes into account the genomic base composition. Finally, we show that RCA outperforms the codon adaptation index (CAI) as a predictor of gene expression when operating on the CAI reference set and that this improvement is significantly larger when analysing genomes with high mutational bias.

Association between mismatch repair gene MSH3 codons 1036 and 222 polymorphisms and sporadic prostate cancer in the Iranian population.

PubMed

Jafary, Fariba; Salehi, Mansoor; Sedghi, Maryam; Nouri, Nayereh; Jafary, Farzaneh; Sadeghi, Farzaneh; Motamedi, Shima; Talebi, Maede

2012-01-01

The mismatch repair system (MMR) is a post-replicative DNA repair mechanism whose defects can lead to cancer. The MSH3 protein is an essential component of the system. We postulated that MSH3 gene polymorphisms might therefore be associated with prostate cancer (PC). We studied MSH3 codon 222 and MSH3 codon 1036 polymorphisms in a group of Iranian sporadic PC patients. A total of 60 controls and 18 patients were assessed using the polymerase chain reaction and single strand conformational polymorphism. For comparing the genotype frequencies of patients and controls the chi-square test was applied. The obtained result indicated that there was significantly association between G/A genotype of MSH3 codon 222 and G/G genotype of MSH3 codon 1036 with an increased PC risk (P=0.012 and P=0.02 respectively). Our results demonstrated that MSH3 codon 222 and MSH3 codon 1036 polymorphisms may be risk factors for sporadic prostate cancer in the Iranian population.

Purification-Free, Target-Selective Immobilization of a Protein from Cell Lysates.

PubMed

Cha, Jaehyun; Kwon, Inchan

2018-02-27

Protein immobilization has been widely used for laboratory experiments and industrial processes. Preparation of a recombinant protein for immobilization usually requires laborious and expensive purification steps. Here, a novel purification-free, target-selective immobilization technique of a protein from cell lysates is reported. Purification steps are skipped by immobilizing a target protein containing a clickable non-natural amino acid (p-azidophenylalanine) in cell lysates onto alkyne-functionalized solid supports via bioorthogonal azide-alkyne cycloaddition. In order to achieve a target protein-selective immobilization, p-azidophenylalanine was introduced into an exogenous target protein, but not into endogenous non-target proteins using host cells with amber codon-free genomic DNAs. Immobilization of superfolder fluorescent protein (sfGFP) from cell lysates is as efficient as that of the purified sfGFP. Using two fluorescent proteins (sfGFP and mCherry), the authors also demonstrated that the target proteins are immobilized with a minimal immobilization of non-target proteins (target-selective immobilization). © 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Reducing codon redundancy and screening effort of combinatorial protein libraries created by saturation mutagenesis.

PubMed

Kille, Sabrina; Acevedo-Rocha, Carlos G; Parra, Loreto P; Zhang, Zhi-Gang; Opperman, Diederik J; Reetz, Manfred T; Acevedo, Juan Pablo

2013-02-15

Saturation mutagenesis probes define sections of the vast protein sequence space. However, even if randomization is limited this way, the combinatorial numbers problem is severe. Because diversity is created at the codon level, codon redundancy is a crucial factor determining the necessary effort for library screening. Additionally, due to the probabilistic nature of the sampling process, oversampling is required to ensure library completeness as well as a high probability to encounter all unique variants. Our trick employs a special mixture of three primers, creating a degeneracy of 22 unique codons coding for the 20 canonical amino acids. Therefore, codon redundancy and subsequent screening effort is significantly reduced, and a balanced distribution of codon per amino acid is achieved, as demonstrated exemplarily for a library of cyclohexanone monooxygenase. We show that this strategy is suitable for any saturation mutagenesis methodology to generate less-redundant libraries.

Diverse expression levels of two codon-optimized genes that encode human papilloma virus type 16 major protein L1 in Hansenula polymorpha.

PubMed

Liu, Cunbao; Yang, Xu; Yao, Yufeng; Huang, Weiwei; Sun, Wenjia; Ma, Yanbing

2014-05-01

Two versions of an optimized gene that encodes human papilloma virus type 16 major protein L1 were designed according to the codon usage frequency of Pichia pastoris. Y16 was highly expressed in both P. pastoris and Hansenula polymorpha. M16 expression was as efficient as that of Y16 in P. pastoris, but merely detectable in H. polymorpha even though transcription levels of M16 and Y16 were similar. H. polymorpha had a unique codon usage frequency that contains many more rare codons than Saccharomyces cerevisiae or P. pastoris. These findings indicate that even codon-optimized genes that are expressed well in S. cerevisiae and P. pastoris may be inefficiently expressed in H. polymorpha; thus rare codons must be avoided when universal optimized gene versions are designed to facilitate expression in a variety of yeast expression systems, especially H. polymorpha is involved.

Evolutionary conservation of codon optimality reveals hidden signatures of cotranslational folding.

PubMed

Pechmann, Sebastian; Frydman, Judith

2013-02-01

The choice of codons can influence local translation kinetics during protein synthesis. Whether codon preference is linked to cotranslational regulation of polypeptide folding remains unclear. Here, we derive a revised translational efficiency scale that incorporates the competition between tRNA supply and demand. Applying this scale to ten closely related yeast species, we uncover the evolutionary conservation of codon optimality in eukaryotes. This analysis reveals universal patterns of conserved optimal and nonoptimal codons, often in clusters, which associate with the secondary structure of the translated polypeptides independent of the levels of expression. Our analysis suggests an evolved function for codon optimality in regulating the rhythm of elongation to facilitate cotranslational polypeptide folding, beyond its previously proposed role of adapting to the cost of expression. These findings establish how mRNA sequences are generally under selection to optimize the cotranslational folding of corresponding polypeptides.

Heterologous expression of proteins from Plasmodium falciparum: results from 1000 genes.

PubMed

Mehlin, Christopher; Boni, Erica; Buckner, Frederick S; Engel, Linnea; Feist, Tiffany; Gelb, Michael H; Haji, Lutfiyah; Kim, David; Liu, Colleen; Mueller, Natascha; Myler, Peter J; Reddy, J T; Sampson, Joshua N; Subramanian, E; Van Voorhis, Wesley C; Worthey, Elizabeth; Zucker, Frank; Hol, Wim G J

2006-08-01

As part of a structural genomics initiative, 1000 open reading frames from Plasmodium falciparum, the causative agent of the most deadly form of malaria, were tested in an E. coli protein expression system. Three hundred and thirty-seven of these targets were observed to express, although typically the protein was insoluble. Sixty-three of the targets provided soluble protein in yields ranging from 0.9 to 406.6 mg from one liter of rich media. Higher molecular weight, greater protein disorder (segmental analysis, SEG), more basic isoelectric point (pI), and a lack of homology to E. coli proteins were all highly and independently correlated with difficulties in expression. Surprisingly, codon usage and the percentage of adenosines and thymidines (%AT) did not appear to play a significant role. Of those proteins which expressed, high pI and a hypothetical annotation were both strongly and independently correlated with insolubility. The overwhelmingly important role of pI in both expression and solubility appears to be a surprising and fundamental issue in the heterologous expression of P. falciparum proteins in E. coli. Twelve targets which did not express in E. coli from the native gene sequence were codon-optimized through whole gene synthesis, resulting in the (insoluble) expression of three of these proteins. Seventeen targets which were expressed insolubly in E. coli were moved into a baculovirus/Sf-21 system, resulting in the soluble expression of one protein at a high level and six others at a low level. A variety of factors conspire to make the heterologous expression of P. falciparum proteins challenging, and these observations lay the groundwork for a rational approach to prioritizing and, ultimately, eliminating these impediments.

Codon usage bias and phylogenetic analysis of mitochondrial ND1 gene in pisces, aves, and mammals.

PubMed

Uddin, Arif; Choudhury, Monisha Nath; Chakraborty, Supriyo

2018-01-01

The mitochondrially encoded NADH:ubiquinone oxidoreductase core subunit 1 (MT-ND1) gene is a subunit of the respiratory chain complex I and involved in the first step of the electron transport chain of oxidative phosphorylation (OXPHOS). To understand the pattern of compositional properties, codon usage and expression level of mitochondrial ND1 genes in pisces, aves, and mammals, we used bioinformatic approaches as no work was reported earlier. In this study, a perl script was used for calculating nucleotide contents and different codon usage bias parameters. The codon usage bias of MT-ND1 was low but the expression level was high as revealed from high ENC and CAI value. Correspondence analysis (COA) suggests that the pattern of codon usage for MT-ND1 gene is not same across species and that compositional constraint played an important role in codon usage pattern of this gene among pisces, aves, and mammals. From the regression equation of GC12 on GC3, it can be inferred that the natural selection might have played a dominant role while mutation pressure played a minor role in influencing the codon usage patterns. Further, ND1 gene has a discrepancy with cytochrome B (CYB) gene in preference of codons as evident from COA. The codon usage bias was low. It is influenced by nucleotide composition, natural selection, mutation pressure, length (number) of amino acids, and relative dinucleotide composition. This study helps in understanding the molecular biology, genetics, evolution of MT-ND1 gene, and also for designing a synthetic gene.

Codon usage and expression level of human mitochondrial 13 protein coding genes across six continents.

PubMed

Chakraborty, Supriyo; Uddin, Arif; Mazumder, Tarikul Huda; Choudhury, Monisha Nath; Malakar, Arup Kumar; Paul, Prosenjit; Halder, Binata; Deka, Himangshu; Mazumder, Gulshana Akthar; Barbhuiya, Riazul Ahmed; Barbhuiya, Masuk Ahmed; Devi, Warepam Jesmi

2017-12-02

The study of codon usage coupled with phylogenetic analysis is an important tool to understand the genetic and evolutionary relationship of a gene. The 13 protein coding genes of human mitochondria are involved in electron transport chain for the generation of energy currency (ATP). However, no work has yet been reported on the codon usage of the mitochondrial protein coding genes across six continents. To understand the patterns of codon usage in mitochondrial genes across six different continents, we used bioinformatic analyses to analyze the protein coding genes. The codon usage bias was low as revealed from high ENC value. Correlation between codon usage and GC3 suggested that all the codons ending with G/C were positively correlated with GC3 but vice versa for A/T ending codons with the exception of ND4L and ND5 genes. Neutrality plot revealed that for the genes ATP6, COI, COIII, CYB, ND4 and ND4L, natural selection might have played a major role while mutation pressure might have played a dominant role in the codon usage bias of ATP8, COII, ND1, ND2, ND3, ND5 and ND6 genes. Phylogenetic analysis indicated that evolutionary relationships in each of 13 protein coding genes of human mitochondria were different across six continents and further suggested that geographical distance was an important factor for the origin and evolution of 13 protein coding genes of human mitochondria. Copyright © 2017 Elsevier B.V. and Mitochondria Research Society. All rights reserved.

Overcoming codon bias: a method for high-level overexpression of Plasmodium and other AT-rich parasite genes in Escherichia coli.

PubMed

Baca, A M; Hol, W G

2000-02-01

Parasite genes often use codons which are rarely used in the highly expressed genes of Escherichia coli, possibly resulting in translational stalling and lower yields of recombinant protein. We have constructed the "RIG" plasmid to overcome the potential codon-bias problem seen in Plasmodium genes. RIG contains the genes that encode three tRNAs (Arg, Ile, Gly), which recognise rare codons found in parasite genes. When co-transformed into E. coli along with expression plasmids containing parasite genes, RIG can greatly increase levels of overexpressed protein. Codon frequency analysis suggests that RIG may be applied to a variety of protozoan and helminth genes.

A Novel Frameshift Mutation at Codons 138/139 (HBB: c.417_418insT) on the β-Globin Gene Leads to β-Thalassemia.

PubMed

Jiang, Fan; Huang, Lv-Yin; Chen, Gui-Lan; Zhou, Jian-Ying; Xie, Xing-Mei; Li, Dong-Zhi

2017-01-01

We describe a new β-thalassemic mutation in a Chinese subject. This allele develops by insertion of one nucleotide (+T) between codons 138 and 139 in the third exon of the β-globin gene. The mutation causes a frameshift that leads to a termination codon at codon 139. In the heterozygote, this allele has the phenotype of classical β-thalassemia (β-thal) minor.

Do multiple herbivores maintain chemical diversity of Scots pine monoterpenes?

PubMed Central

Iason, Glenn R.; O'Reilly-Wapstra, Julianne M.; Brewer, Mark J.; Summers, Ron W.; Moore, Ben D.

2011-01-01

A central issue in our understanding of the evolution of the diversity of plant secondary metabolites (PSMs) is whether or not compounds are functional, conferring an advantage to the plant, or non-functional. We examine the hypothesis that the diversity of monoterpene PSMs within a plant species (Scots pine Pinus sylvestris) may be explained by different compounds acting as defences against high-impact herbivores operating at different life stages. We also hypothesize that pairwise coevolution, with uncorrelated interactions, is more likely to result in greater PSM diversity, than diffuse coevolution. We tested whether up to 13 different monoterpenes in Scots pine were inhibitory to herbivory by slugs (Arion ater), bank voles (Clethrionomys glareolus), red deer (Cervus elaphus) and capercaillie (Tetrao urogallus), each of which attack trees at a different life stage. Plants containing more α-pinene were avoided by both slugs and capercaillie, which may act as reinforcing selective agents for this dominant defensive compound. Herbivory by red deer and capercaillie were, respectively, weakly negatively associated with δ3-carene, and strongly negatively correlated with the minor compound β-ocimene. Three of the four herbivores are probably contributory selective agents on some of the terpenes, and thus maintain some, but by no means all, of the phytochemical diversity in the species. The correlated defensive function of α-pinene against slugs and capercaillie is consistent with diffuse coevolutionary processes. PMID:21444308

Ketone body metabolism and its defects.

PubMed

Fukao, Toshiyuki; Mitchell, Grant; Sass, Jörn Oliver; Hori, Tomohiro; Orii, Kenji; Aoyama, Yuka

2014-07-01

Acetoacetate (AcAc) and 3-hydroxybutyrate (3HB), the two main ketone bodies of humans, are important vectors of energy transport from the liver to extrahepatic tissues, especially during fasting, when glucose supply is low. Blood total ketone body (TKB) levels should be evaluated in the context of clinical history, such as fasting time and ketogenic stresses. Blood TKB should also be evaluated in parallel with blood glucose and free fatty acids (FFA). The FFA/TKB ratio is especially useful for evaluation of ketone body metabolism. Defects in ketogenesis include mitochondrial HMG-CoA synthase (mHS) deficiency and HMG-CoA lyase (HL) deficiency. mHS deficiency should be considered in non-ketotic hypoglycemia if a fatty acid beta-oxidation defect is suspected, but cannot be confirmed. Patients with HL deficiency can develop hypoglycemic crises and neurological symptoms even in adolescents and adults. Succinyl-CoA-3-oxoacid CoA transferase (SCOT) deficiency and beta-ketothiolase (T2) deficiency are two defects in ketolysis. Permanent ketosis is pathognomonic for SCOT deficiency. However, patients with "mild" SCOT mutations may have nonketotic periods. T2-deficient patients with "mild" mutations may have normal blood acylcarnitine profiles even in ketoacidotic crises. T2 deficient patients cannot be detected in a reliable manner by newborn screening using acylcarnitines. We review recent data on clinical presentation, metabolite profiles and the course of these diseases in adults, including in pregnancy.

Hydraulic adjustment of Scots pine across Europe.

PubMed

Martínez-Vilalta, J; Cochard, H; Mencuccini, M; Sterck, F; Herrero, A; Korhonen, J F J; Llorens, P; Nikinmaa, E; Nolè, A; Poyatos, R; Ripullone, F; Sass-Klaassen, U; Zweifel, R

2009-10-01

* The variability of branch-level hydraulic properties was assessed across 12 Scots pine populations covering a wide range of environmental conditions, including some of the southernmost populations of the species. The aims were to relate this variability to differences in climate, and to study the potential tradeoffs between traits. * Traits measured included wood density, radial growth, xylem anatomy, sapwood- and leaf-specific hydraulic conductivity (K(S) and K(L)), vulnerability to embolism, leaf-to-sapwood area ratio (A(L) : A(S)), needle carbon isotope discrimination (Delta13C) and nitrogen content, and specific leaf area. * Between-population variability was high for most of the hydraulic traits studied, but it was directly associated with climate dryness (defined as a combination of atmospheric moisture demand and availability) only for A(L) : A(S), K(L) and Delta13C. Shoot radial growth and A(L) : A(S) declined with stand development, which is consistent with a strategy to avoid exceedingly low water potentials as tree size increases. In addition, we did not find evidence at the intraspecific level of some associations between hydraulic traits that have been commonly reported across species. * The adjustment of Scots pine's hydraulic system to local climatic conditions occurred primarily through modifications of A(L) : A(S) and direct stomatal control, whereas intraspecific variation in vulnerability to embolism and leaf physiology appears to be limited.

Variation and inheritance pattern in cone and seed characteristics of Scots pine (Pinus sylvestris L.) for evaluation of genetic diversity.

PubMed

Sevik, Hakan; Topaçoğlu, Osman

2015-09-01

Scots pine (Pinus sylvestris L.) is one of the most common and important forest tree species in Turkey due to usefulness of its wood to many commercial uses. This species is classified as one of the economically important tree species for Turkish Forestry in the "National Tree Breeding and Seed Production Program". The objective of the present study was to investigate variation and inheritance pattern in cone and seed characteristics of Scots pine and to evaluate variation in cone and seed characters within and among clones and grafts. The results showed that maximum CV among the clones was found for SWe (21.95), FS (16.99) and CWe (16.88). According to the results of SAS, variation between the clones is averaged at 19.2% and variation within the clones is averaged at 24.4 %. Variation between the clones ranged from 3.6% (SW) to 34.5% (TC) and variation within the clones ranged from 12.3% (SW) to 38.1% (WL). For CW, AL, AW, WW and TC, genetic variation among clones was higher than within clones. When the results of study like compared with results obtained from natural populations, it was seen that genetic variability in seed orchard which was subjected to study was quite low. This case may have dangerous results for the future of forests.

A UV resonance Raman (UVRR) spectroscopic study on the extractable compounds in Scots pine (Pinus sylvestris) wood. Part II. Hydrophilic compounds.

PubMed

Nuopponen, M; Willför, S; Jääskeläinen, A-S; Vuorinen, T

2004-11-01

Hydrophilic extracts of Scots pine (Pinus sylvestris) heartwood and sapwood and a solid Scots pine knotwood sample were studied by UV resonance Raman spectroscopy (UVRRS). In addition, UVRR spectra of two hydrophilic model compounds (pinosylvin and chrysin) were analysed. UV Raman spectra were collected using 244 and 257 nm excitation wavelengths. The chemical composition of the acetone:water (95:5 v/v) extracts were also determined by gas chromatography. The aromatic and oleophilic structures of pinosylvin and chrysin showed three intense resonance enhanced bands in the spectral region of 1649-1548 cm(-1). Pinosylvin showed also a relatively intense band in the aromatic substitution region at 996 cm(-1). The spectra of the heartwood acetone:water extract showed many bands typical of pinosylvin. In addition, the extract included bands distinctive for resin and fatty acids. The sapwood acetone:water extract showed bands due to oleophilic structures at 1655-1650 cm(-1). The extract probably also contained oligomeric lignans because the UVRR spectra were in parts similar to that of guaiacyl lignin. The characteristic band of pinosylvin (996 cm(-1)) was detected in the UVRR spectrum of the resin rich knotwood. In addition, several other bands typical for wood resin were observed, which indicated that the wood resin in the knotwood was resonance enhanced even more than lignin.

A UV resonance Raman (UVRR) spectroscopic study on the extractable compounds in Scots pine ( Pinus sylvestris) wood . Part II. Hydrophilic compounds

NASA Astrophysics Data System (ADS)

Nuopponen, M.; Willför, S.; Jääskeläinen, A.-S.; Vuorinen, T.

2004-11-01

Hydrophilic extracts of Scots pine ( Pinus sylvestris) heartwood and sapwood and a solid Scots pine knotwood sample were studied by UV resonance Raman spectroscopy (UVRRS). In addition, UVRR spectra of two hydrophilic model compounds (pinosylvin and chrysin) were analysed. UV Raman spectra were collected using 244 and 257 nm excitation wavelengths. The chemical composition of the acetone:water (95:5 v/v) extracts were also determined by gas chromatography. The aromatic and oleophilic structures of pinosylvin and chrysin showed three intense resonance enhanced bands in the spectral region of 1649-1548 cm -1. Pinosylvin showed also a relatively intense band in the aromatic substitution region at 996 cm -1. The spectra of the heartwood acetone:water extract showed many bands typical of pinosylvin. In addition, the extract included bands distinctive for resin and fatty acids. The sapwood acetone:water extract showed bands due to oleophilic structures at 1655-1650 cm -1. The extract probably also contained oligomeric lignans because the UVRR spectra were in parts similar to that of guaiacyl lignin. The characteristic band of pinosylvin (996 cm -1) was detected in the UVRR spectrum of the resin rich knotwood. In addition, several other bands typical for wood resin were observed, which indicated that the wood resin in the knotwood was resonance enhanced even more than lignin.

Comparing the VOC emissions between air-dried and heat-treated Scots pine wood

NASA Astrophysics Data System (ADS)

Manninen, Anne-Marja; Pasanen, Pertti; Holopainen, Jarmo K.

The emissions of volatile organic compounds (VOCs) from air-dried Scots pine wood and from heat-treated Scots pine wood were compared with GC-MS analysis. Air-dried wood blocks released about 8 times more total VOCs than heat-treated (24 h at 230°C) ones. Terpenes were clearly the main compound group in the air-dried wood samples, whereas aldehydes and carboxylic acids and their esters dominated in the heat-treated wood samples. Only 14 compounds out of 41 identified individual compounds were found in both wood samples indicating considerable changes in VOC emission profile during heat-treatment process. Of individual compounds α-pinene, 3-carene and hexanal were the most abundant ones in the air-dried wood. By contrast, in the heat-treated wood 2-furancarboxaldehyde, acetic acid and 2-propanone were the major compounds of VOC emission. Current emission results reveal that significant chemical changes have occurred, and volatile monoterpenes and other low-molecular-weight compounds have evaporated from the wood during the heat-treatment process when compared to air-dried wood. Major chemical changes detected in VOC emissions are explained by the thermal degradation and oxidation of main constituents in wood. The results suggest that if heat-treated wood is used in interior carpentry, emissions of monoterpenes are reduced compared to air-dried wood, but some irritating compounds might be released into indoor air.

Frost hardiness of mycorrhizal (Hebeloma sp.) and non-mycorrhizal Scots pine roots.

PubMed

Korhonen, Anna; Lehto, Tarja; Repo, Tapani

2013-10-01

The frost hardiness (FH) of mycorrhizal [ectomycorrhizal (ECM)] and non-mycorrhizal (NM) Scots pine (Pinus sylvestris) seedlings was studied to assess whether mycorrhizal symbiosis affected the roots' tolerance of below-zero temperatures. ECM (Hebeloma sp.) and NM seedlings were cultivated in a growth chamber for 18 weeks. After 13 weeks' growth in long-day and high-temperature (LDHT) conditions, a half of the ECM and NM seedlings were moved into a chamber with short-day and low-temperature (SDLT) conditions to cold acclimate. After exposures to a range of below-zero temperatures, the FH of the roots was assessed by means of the relative electrolyte leakage test. The FH was determined as the inflection point of the temperature-response curve. No significant difference was found between the FH of mycorrhizal and non-mycorrhizal roots in LDHT (-8.9 and -9.8 °C) or SDLT (-7.5 and -6.8 °C). The mycorrhizal treatment had no significant effect on the total dry mass, the allocation of dry mass among the roots and needles or nutrient accumulation. The mycorrhizal treatment with Hebeloma sp. did not affect the FH of Scots pine in this experimental setup. More information is needed on the extent to which mycorrhizas tolerate low temperatures, especially with different nutrient contents and different mycorrhiza fungi.

Are Scots pine forest edges particularly prone to drought-induced mortality?

NASA Astrophysics Data System (ADS)

Buras, Allan; Schunk, Christian; Zeiträg, Claudia; Herrmann, Corinna; Kaiser, Laura; Lemme, Hannes; Straub, Christoph; Taeger, Steffen; Gößwein, Sebastian; Klemmt, Hans-Joachim; Menzel, Annette

2018-02-01

Climate change is expected to exacerbate the frequency of drought-induced tree mortality world-wide. To better predict the associated change of species composition and forest dynamics on various scales and develop adequate adaptation strategies, more information on the mechanisms driving the often observed patchiness of tree die-back is needed. Although forest-edge effects may play an important role within the given context, only few corresponding studies exist. Here, we investigate the regional die-back of Scots pine in Franconia, Germany, after a hot and dry summer in 2015, thereby emphasizing possible differences in mortality between forest edge and interior. By means of dendroecological investigations and close-range remote sensing, we assess long-term growth performance and current tree vitality along five different forest-edge distance gradients. Our results clearly indicate a differing growth performance between edge and interior trees, associated with a higher vulnerability to drought, increased mortality rates, and lower tree vitality at the forest edge. Prior long-lasting growth decline of dead trees compared to live trees suggests depletion of carbon reserves in course of a long-term drought persisting since the 1990s to be the cause of regional Scots pine die-back. These findings highlight the forest edge as a potential focal point of forest management adaptation strategies in the context of drought-induced mortality.

The history of mercury pollution near the Spolana chlor-alkali plant (Neratovice, Czech Republic) as recorded by Scots pine tree rings and other bioindicators

USGS Publications Warehouse

Navrátil, Tomáš; Šimeček, Martin; Shanley, James B.; Rohovec, Jan; Hojdová, Maria; Houška, Jakub

2017-01-01

We assessed > 100 years of mercury (Hg) pollution recorded in the tree rings of Scots Pine near a Czech chlor-alkali plant operating since 1941. Hg concentrations in tree rings increased with the launching of plant operations and decreased when Hg emissions decreased in 1975 due to an upgrade in production technology. Similar to traditional bioindicators of pollution such as pine needles, bark and forest floor humus, Hg concentrations in Scots Pine boles decreased with distance from the plant. Mean Hg in pine bole in the 1940s ranged from 32.5 μg/kg Hg at a distance of 0.5 km from the plant to 5.4 μg/kg at a distance of > 4.7 km, where tree ring Hg was the same as at a reference site, and other bioindicators also suggest that the effect of the plant was no longer discernible. Tree ring Hg concentrations decreased by 8–29 μg/kg since the 1940s at all study sites including the reference site. The lack of exact correspondence between changes at the plant and tree ring Hg indicated some smearing of the signal due to lateral translocation of Hg from sapwood to heartwood. Bole Hg concentrations reflected local and regional atmospheric Hg concentrations, and not Hg wet deposition.

Darwin Assembly: fast, efficient, multi-site bespoke mutagenesis

PubMed Central

Cozens, Christopher

2018-01-01

Abstract Engineering proteins for designer functions and biotechnological applications almost invariably requires (or at least benefits from) multiple mutations to non-contiguous residues. Several methods for multiple site-directed mutagenesis exist, but there remains a need for fast and simple methods to efficiently introduce such mutations – particularly for generating large, high quality libraries for directed evolution. Here, we present Darwin Assembly, which can deliver high quality libraries of >108 transformants, targeting multiple (>10) distal sites with minimal wild-type contamination (<0.25% of total population) and which takes a single working day from purified plasmid to library transformation. We demonstrate its efficacy with whole gene codon reassignment of chloramphenicol acetyl transferase, mutating 19 codons in a single reaction in KOD DNA polymerase and generating high quality, multiple-site libraries in T7 RNA polymerase and Tgo DNA polymerase. Darwin Assembly uses commercially available enzymes, can be readily automated, and offers a cost-effective route to highly complex and customizable library generation. PMID:29409059

Methyl Jasmonate-Induced Monoterpenes in Scots Pine and Norway Spruce Tissues Affect Pine Weevil Orientation.

PubMed

Lundborg, Lina; Nordlander, Göran; Björklund, Niklas; Nordenhem, Henrik; Borg-Karlson, Anna-Karin

2016-12-01

In large parts of Europe, insecticide-free measures for protecting conifer plants are desired to suppress damage by the pine weevil Hylobius abietis (L.). Treatment with methyl jasmonate (MeJA), a chemical elicitor already used in crop production, may enhance expression of chemical defenses in seedlings in conifer regenerations. However, in a previous experiment, MeJA treatment resulted in substantially better field protection for Scots pine (Pinus sylvestris L.) than for Norway spruce (Picea abies (L.) Karst.). Hypothesizing that the variations may be at least due partly to volatiles released by MeJA-treated seedlings and their effects on pine weevil orientation, we examined tissue extracts of seedlings (from the same batches as previously used) by two-dimensional GC-MS. We found that the MeJA treatment increased contents of the monoterpene (-)-β-pinene in phloem (the weevil's main target tissue) of both tree species, however, the (-)-β-pinene/(-)-α-pinene ratio increased more in the phloem of P. sylvestris. We also tested the attractiveness of individual monoterpenes found in conifer tissues (needles and phloem) for pine weevils using an arena with traps baited with single-substance dispensers and pine twigs. Trap catches were reduced when the pine material was combined with a dispenser releasing (-)-β-pinene, (+)-3-carene, (-)-bornyl acetate or 1,8-cineole. However, (-)-α-pinene did not have this effect. Thus, the greater field protection of MeJA-treated P. sylvestris seedlings may be due to the selective induction of increases in contents of the deterrent (-)-β-pinene, in contrast to strong increases in both non-deterrent (-)-α-pinene and the deterrent (-)-β-pinene in P. abies seedlings.

Influence of certain forces on evolution of synonymous codon usage bias in certain species of three basal orders of aquatic insects.

PubMed

Selva Kumar, C; Nair, Rahul R; Sivaramakrishnan, K G; Ganesh, D; Janarthanan, S; Arunachalam, M; Sivaruban, T

2012-12-01

Forces that influence the evolution of synonymous codon usage bias are analyzed in six species of three basal orders of aquatic insects. The rationale behind choosing six species of aquatic insects (three from Ephemeroptera, one from Plecoptera, and two from Odonata) for the present analysis is based on phylogenetic position at the basal clades of the Order Insecta facilitating the understanding of the evolution of codon bias and of factors shaping codon usage patterns in primitive clades of insect lineages and their subtle differences in some of their ecological and environmental requirements in terms of habitat-microhabitat requirements, altitudinal preferences, temperature tolerance ranges, and consequent responses to climate change impacts. The present analysis focuses on open reading frames of the 13 protein-coding genes in the mitochondrial genome of six carefully chosen insect species to get a comprehensive picture of the evolutionary intricacies of codon bias. In all the six species, A and T contents are observed to be significantly higher than G and C, and are used roughly equally. Since transcription hypothesis on codon usage demands A richness and T poorness, it is quite likely that mutation pressure may be the key factor associated with synonymous codon usage (SCU) variations in these species because the mutation hypothesis predicts AT richness and GC poorness in the mitochondrial DNA. Thus, AT-biased mutation pressure seems to be an important factor in framing the SCU variation in all the selected species of aquatic insects, which in turn explains the predominance of A and T ending codons in these species. This study does not find any association between microhabitats and codon usage variations in the mitochondria of selected aquatic insects. However, this study has identified major forces, such as compositional constraints and mutation pressure, which shape patterns of codon usage in mitochondrial genes in the primitive clades of insect lineages.

Three stages in the evolution of the genetic code

NASA Technical Reports Server (NTRS)

Baumann, U.; Oro, J.

1993-01-01

A diversification of the genetic code based on the number of codons available for the proteinous amino acids is established. Three groups of amino acids during evolution of the code are distinguished. On the basis of their chemical complexity those amino acids emerging later in a translation process are derived. Codon number and chemical complexity indicate that His, Phe, Tyr, Cys and either Lys or Asn were introduced in the second stage, whereas the number of codons alone gives evidence that Trp and Met were introduced in the third stage. The amino acids of stage 1 use purine-rich codons, while all the amino acids introduced in the second stage, in contrast, use pyrimidines in the third position of their codons. A low abundance of pyrimidines during early translation is derived. This assumption is supported by experiments on non-enzymatic replication and interactions of hairpin loops with a complementary strand. A back extrapolation concludes a high purine content of the first nucleic acids, which gradually decreased during their evolution. Amino acids independently available from prebiotic synthesis were thus correlated to purine-rich codons. Implications on the prebiotic replication are discussed also in the light of recent codon usage data.

Identification and codon reading properties of 5-cyanomethyl uridine, a new modified nucleoside found in the anticodon wobble position of mutant haloarchaeal isoleucine tRNAs

PubMed Central

Mandal, Debabrata; Köhrer, Caroline; Su, Dan; Babu, I. Ramesh; Chan, Clement T.Y.; Liu, Yuchen; Söll, Dieter; Blum, Paul; Kuwahara, Masayasu; Dedon, Peter C.; RajBhandary, Uttam L.

2014-01-01

Most archaea and bacteria use a modified C in the anticodon wobble position of isoleucine tRNA to base pair with A but not with G of the mRNA. This allows the tRNA to read the isoleucine codon AUA without also reading the methionine codon AUG. To understand why a modified C, and not U or modified U, is used to base pair with A, we mutated the C34 in the anticodon of Haloarcula marismortui isoleucine tRNA (tRNA2Ile) to U, expressed the mutant tRNA in Haloferax volcanii, and purified and analyzed the tRNA. Ribosome binding experiments show that although the wild-type tRNA2Ile binds exclusively to the isoleucine codon AUA, the mutant tRNA binds not only to AUA but also to AUU, another isoleucine codon, and to AUG, a methionine codon. The G34 to U mutant in the anticodon of another H. marismortui isoleucine tRNA species showed similar codon binding properties. Binding of the mutant tRNA to AUG could lead to misreading of the AUG codon and insertion of isoleucine in place of methionine. This result would explain why most archaea and bacteria do not normally use U or a modified U in the anticodon wobble position of isoleucine tRNA for reading the codon AUA. Biochemical and mass spectrometric analyses of the mutant tRNAs have led to the discovery of a new modified nucleoside, 5-cyanomethyl U in the anticodon wobble position of the mutant tRNAs. 5-Cyanomethyl U is present in total tRNAs from euryarchaea but not in crenarchaea, eubacteria, or eukaryotes. PMID:24344322

The complete mitochondrial genome of Setaria digitata (Nematoda: Filarioidea): Mitochondrial gene content, arrangement and composition compared with other nematodes.

PubMed

Yatawara, Lalani; Wickramasinghe, Susiji; Rajapakse, R P V J; Agatsuma, Takeshi

2010-09-01

In the present study, we determined the complete mitochondrial (mt) genome sequence (13,839bp) of parasitic nematode Setaria digitata and its structure and organization compared with Onchocerca volvulus, Dirofilaria immitis and Brugia malayi. The mt genome of S. digitata is slightly larger than the mt genomes of other filarial nematodes. S. digitata mt genome contains 36 genes (12 protein-coding genes, 22 transfer RNAs and 2 ribosomal RNAs) that are typically found in metazoans. This genome contains a high A+T (75.1%) content and low G+C content (24.9%). The mt gene order for S. digitata is the same as those for O. volvulus, D. immitis and B. malayi but it is distinctly different from other nematodes compared. The start codons inferred in the mt genome of S. digitata are TTT, ATT, TTG, ATG, GTT and ATA. Interestingly, the initiation codon TTT is unique to S. digitata mt genome and four protein-coding genes use this codon as a translation initiation codon. Five protein-coding genes use TAG as a stop codon whereas three genes use TAA and four genes use T as a termination codon. Out of 64 possible codons, only 57 are used for mitochondrial protein-coding genes of S. digitata. T-rich codons such as TTT (18.9%), GTT (7.9%), TTG (7.8%), TAT (7%), ATT (5.7%), TCT (4.8%) and TTA (4.1%) are used more frequently. This pattern of codon usage reflects the strong bias for T in the mt genome of S. digitata. In conclusion, the present investigation provides new molecular data for future studies of the comparative mitochondrial genomics and systematic of parasitic nematodes of socio-economic importance. 2010 Elsevier B.V. All rights reserved.

Demonstration of GTG as an endogenous initiation codon for a human mRNA transcript revealed by molecular cloning of the serpin endopin 2B.

PubMed

Hwang, Shin-Rong; Garza, Christina Z; Wegrzyn, Jill; Hook, Vivian Y H

2004-08-16

This study demonstrates utilization of the novel GTG initiation codon for translation of a human mRNA transcript that encodes the serpin endopin 2B, a protease inhibitor. Molecular cloning revealed the nucleotide sequence of the human endopin 2B cDNA. Its deduced primary sequence shows high homology to bovine endopin 2A that possesses cross-class protease inhibition of elastase and papain. Notably, the human endopin 2B cDNA sequence revealed GTG as the predicted translation initiation codon; the predicted translation product of 46 kDa endopin 2B was produced by in vitro translation of 35S-endopin 2B with mammalian (rabbit) protein translation components. Importantly, bioinformatic studies demonstrated the presence of the entire human endopin 2B cDNA sequence with GTG as initiation codon within the human genome on chromosome 14. Further evidence for GTG as a functional initiation codon was illustrated by GTG-mediated in vitro translation of the heterologous protein EGFP, and by GTG-mediated expression of EGFP in mammalian PC12 cells. Mutagenesis of GTG to GTC resulted in the absence of EGFP expression in PC12 cells, indicating the function of GTG as an initiation codon. In addition, it was apparent that the GTG initiation codon produces lower levels of translated protein compared to ATG as initiation codon. Significantly, GTG-mediated translation of endopin 2B demonstrates a functional human gene product not previously predicted from initial analyses of the human genome. Further analyses based on GTG as an alternative initiation codon may predict new candidate genes of the human genome.

Near-cognate suppression of amber, opal and quadruplet codons competes with aminoacyl-tRNAPyl for genetic code expansion

PubMed Central

O’Donoghue, Patrick; Prat, Laure; Heinemann, Ilka U.; Ling, Jiqiang; Odoi, Keturah; Liu, Wenshe R.; Söll, Dieter

2012-01-01

Over 300 amino acids are found in proteins in nature, yet typically only 20 are genetically encoded. Reassigning stop codons and use of quadruplet codons emerged as the main avenues for genetically encoding non-canonical amino acids (NCAAs). Canonical aminoacyl-tRNAs with near-cognate anticodons also read these codons to some extent. This background suppression leads to ‘statistical protein’ that contains some natural amino acid(s) at a site intended for NCAA. We characterize near-cognate suppression of amber, opal and a quadruplet codon in common Escherichia coli laboratory strains and find that the PylRS/tRNAPyl orthogonal pair cannot completely outcompete contamination by natural amino acids. PMID:23036644

Pre-trial inter-laboratory analytical validation of the FOCUS4 personalised therapy trial.

PubMed

Richman, Susan D; Adams, Richard; Quirke, Phil; Butler, Rachel; Hemmings, Gemma; Chambers, Phil; Roberts, Helen; James, Michelle D; Wozniak, Sue; Bathia, Riya; Pugh, Cheryl; Maughan, Timothy; Jasani, Bharat

2016-01-01

Molecular characterisation of tumours is increasing personalisation of cancer therapy, tailored to an individual and their cancer. FOCUS4 is a molecularly stratified clinical trial for patients with advanced colorectal cancer. During an initial 16-week period of standard first-line chemotherapy, tumour tissue will undergo several molecular assays, with the results used for cohort allocation, then randomisation. Laboratories in Leeds and Cardiff will perform the molecular testing. The results of a rigorous pre-trial inter-laboratory analytical validation are presented and discussed. Wales Cancer Bank supplied FFPE tumour blocks from 97 mCRC patients with consent for use in further research. Both laboratories processed each sample according to an agreed definitive FOCUS4 laboratory protocol, reporting results directly to the MRC Trial Management Group for independent cross-referencing. Pyrosequencing analysis of mutation status at KRAS codons12/13/61/146, NRAS codons12/13/61, BRAF codon600 and PIK3CA codons542/545/546/1047, generated highly concordant results. Two samples gave discrepant results; in one a PIK3CA mutation was detected only in Leeds, and in the other, a PIK3CA mutation was only detected in Cardiff. pTEN and mismatch repair (MMR) protein expression was assessed by immunohistochemistry (IHC) resulting in 6/97 discordant results for pTEN and 5/388 for MMR, resolved upon joint review. Tumour heterogeneity was likely responsible for pyrosequencing discrepancies. The presence of signet-ring cells, necrosis, mucin, edge-effects and over-counterstaining influenced IHC discrepancies. Pre-trial assay analytical validation is essential to ensure appropriate selection of patients for targeted therapies. This is feasible for both mutation testing and immunohistochemical assays and must be built into the workup of such trials. ISRCTN90061564. Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://www.bmj.com/company/products-services/rights-and-licensing/

Importance of codon usage for the temporal regulation of viral gene expression

PubMed Central

Shin, Young C.; Bischof, Georg F.; Lauer, William A.; Desrosiers, Ronald C.

2015-01-01

The glycoproteins of herpesviruses and of HIV/SIV are made late in the replication cycle and are derived from transcripts that use an unusual codon usage that is quite different from that of the host cell. Here we show that the actions of natural transinducers from these two different families of persistent viruses (Rev of SIV and ORF57 of the rhesus monkey rhadinovirus) are dependent on the nature of the skewed codon usage. In fact, the transinducibility of expression of these glycoproteins by Rev and by ORF57 can be flipped simply by changing the nature of the codon usage. Even expression of a luciferase reporter could be made Rev dependent or ORF57 dependent by distinctive changes to its codon usage. Our findings point to a new general principle in which different families of persisting viruses use a poor codon usage that is skewed in a distinctive way to temporally regulate late expression of structural gene products. PMID:26504241

Comparison of codon usage bias across Leishmania and Trypanosomatids to understand mRNA secondary structure, relative protein abundance and pathway functions.

PubMed

Subramanian, Abhishek; Sarkar, Ram Rup

2015-10-01

Understanding the variations in gene organization and its effect on the phenotype across different Leishmania species, and to study differential clinical manifestations of parasite within the host, we performed large scale analysis of codon usage patterns between Leishmania and other known Trypanosomatid species. We present the causes and consequences of codon usage bias in Leishmania genomes with respect to mutational pressure, translational selection and amino acid composition bias. We establish GC bias at wobble position that governs codon usage bias across Leishmania species, rather than amino acid composition bias. We found that, within Leishmania, homogenous codon context coding for less frequent amino acid pairs and codons avoiding formation of folding structures in mRNA are essentially chosen. We predicted putative differences in global expression between genes belonging to specific pathways across Leishmania. This explains the role of evolution in shaping the otherwise conserved genome to demonstrate species-specific function-level differences for efficient survival. Copyright © 2015 Elsevier Inc. All rights reserved.

Theoretical foundations for quantitative paleogenetics. III - The molecular divergence of nucleic acids and proteins for the case of genetic events of unequal probability

NASA Technical Reports Server (NTRS)

Holmquist, R.; Pearl, D.

1980-01-01

Theoretical equations are derived for molecular divergence with respect to gene and protein structure in the presence of genetic events with unequal probabilities: amino acid and base compositions, the frequencies of nucleotide replacements, the usage of degenerate codons, the distribution of fixed base replacements within codons and the distribution of fixed base replacements among codons. Results are presented in the form of tables relating the probabilities of given numbers of codon base changes with respect to the original codon for the alpha hemoglobin, beta hemoglobin, myoglobin, cytochrome c and parvalbumin group gene families. Application of the calculations to the rabbit alpha and beta hemoglobin mRNAs and proteins indicates that the genes are separated by about 425 fixed based replacements distributed over 114 codon sites, which is a factor of two greater than previous estimates. The theoretical results also suggest that many more base replacements are required to effect a given gene or protein structural change than previously believed.

Expression of codon-optmized phosphoenolpyruvate carboxylase gene from Glaciecola sp. HTCC2999 in Escherichia coli and its application for C4 chemical production.

PubMed

Park, Soohyun; Pack, Seung Pil; Lee, Jinwon

2012-08-01

We examined the expression of the phosphoenolpyruvate carboxylase (PEPC) gene from marine bacteria in Escherichia coli using codon optimization. The codon-optimized PEPC gene was expressed in the E. coli K-12 strain W3110. SDS-PAGE analysis revealed that the codon-optimized PEPC gene was only expressed in E. coli, and measurement of enzyme activity indicated the highest PEPC activity in the E. coli SGJS112 strain that contained the codon-optimized PEPC gene. In fermentation assays, the E. coli SGJS112 produced the highest yield of oxaloacetate using glucose as the source and produced a 20-times increase in the yield of malate compared to the control. We concluded that the codon optimization enabled E. coli to express the PEPC gene derived from the Glaciecola sp. HTCC2999. Also, the expressed protein exhibited an enzymatic activity similar to that of E. coli PEPC and increased the yield of oxaloacetate and malate in an E. coli system.

Stop codon readthrough generates a C-terminally extended variant of the human vitamin D receptor with reduced calcitriol response

PubMed Central

Loughran, Gary; Jungreis, Irwin; Tzani, Ioanna; Power, Michael; Dmitriev, Ruslan I.; Ivanov, Ivaylo P.; Kellis, Manolis; Atkins, John F.

2018-01-01

Although stop codon readthrough is used extensively by viruses to expand their gene expression, verified instances of mammalian readthrough have only recently been uncovered by systems biology and comparative genomics approaches. Previously, our analysis of conserved protein coding signatures that extend beyond annotated stop codons predicted stop codon readthrough of several mammalian genes, all of which have been validated experimentally. Four mRNAs display highly efficient stop codon readthrough, and these mRNAs have a UGA stop codon immediately followed by CUAG (UGA_CUAG) that is conserved throughout vertebrates. Extending on the identification of this readthrough motif, we here investigated stop codon readthrough, using tissue culture reporter assays, for all previously untested human genes containing UGA_CUAG. The readthrough efficiency of the annotated stop codon for the sequence encoding vitamin D receptor (VDR) was 6.7%. It was the highest of those tested but all showed notable levels of readthrough. The VDR is a member of the nuclear receptor superfamily of ligand-inducible transcription factors, and it binds its major ligand, calcitriol, via its C-terminal ligand-binding domain. Readthrough of the annotated VDR mRNA results in a 67 amino acid–long C-terminal extension that generates a VDR proteoform named VDRx. VDRx may form homodimers and heterodimers with VDR but, compared with VDR, VDRx displayed a reduced transcriptional response to calcitriol even in the presence of its partner retinoid X receptor. PMID:29386352

Stop codons in the hepatitis B surface proteins are enriched during antiviral therapy and are associated with host cell apoptosis

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

Colledge, Danielle; Soppe, Sally; Yuen, Lilly

Premature stop codons in the hepatitis B virus (HBV) surface protein can be associated with nucleos(t)ide analogue resistance due to overlap of the HBV surface and polymerase genes. The aim of this study was to determine the effect of the replication of three common surface stop codon variants on the hepatocyte. Cell lines were transfected with infectious HBV clones encoding surface stop codons rtM204I/sW196*, rtA181T/sW172*, rtV191I/sW182*, and a panel of substitutions in the surface proteins. HBsAg was measured by Western blotting. Proliferation and apoptosis were measured using flow cytometry. All three surface stop codon variants were defective in HBsAg secretion.more » Cells transfected with these variants were less proliferative and had higher levels of apoptosis than those transfected with variants that did not encode surface stop codons. The most cytopathic variant was rtM204I/sW196*. Replication of HBV encoding surface stop codons was toxic to the cell and promoted apoptosis, exacerbating disease progression. - Highlights: •Under normal circumstances, HBV replication is not cytopathic. •Premature stop codons in the HBV surface protein can be selected and enriched during nucleos(t)ide analogue therapy. •Replication of these variants can be cytopathic to the cell and promote apoptosis. •Inadequate antiviral therapy may actually promote disease progression.« less

Comparative Genomic Analysis MERS CoV Isolated from Humans and Camels with Special Reference to Virus Encoded Helicase.

PubMed

Alnazawi, Mohamed; Altaher, Abdallah; Kandeel, Mahmoud

2017-01-01

Middle East Respiratory Syndrome Coronavirus (MERS CoV) is a new emerging viral disease characterized by high fatality rate. Understanding MERS CoV genetic aspects and codon usage pattern is important to understand MERS CoV survival, adaptation, evolution, resistance to innate immunity, and help in finding the unique aspects of the virus for future drug discovery experiments. In this work, we provide comprehensive analysis of 238 MERS CoV full genomes comprised of human (hMERS) and camel (cMERS) isolates of the virus. MERS CoV genome shaping seems to be under compositional and mutational bias, as revealed by preference of A/T over G/C nucleotides, preferred codons, nucleotides at the third position of codons (NT3s), relative synonymous codon usage, hydropathicity (Gravy), and aromaticity (Aromo) indices. Effective number of codons (ENc) analysis reveals a general slight codon usage bias. Codon adaptation index reveals incomplete adaptation to host environment. MERS CoV showed high ability to resist the innate immune response by showing lower CpG frequencies. Neutrality evolution analysis revealed a more significant role of mutation pressure in cMERS over hMERS. Correspondence analysis revealed that MERS CoV genomes have three genetic clusters, which were distinct in their codon usage, host, and geographic distribution. Additionally, virtual screening and binding experiments were able to identify three new virus-encoded helicase binding compounds. These compounds can be used for further optimization of inhibitors.

GC-Content of Synonymous Codons Profoundly Influences Amino Acid Usage

PubMed Central

Li, Jing; Zhou, Jun; Wu, Ying; Yang, Sihai; Tian, Dacheng

2015-01-01

Amino acids typically are encoded by multiple synonymous codons that are not used with the same frequency. Codon usage bias has drawn considerable attention, and several explanations have been offered, including variation in GC-content between species. Focusing on a simple parameter—combined GC proportion of all the synonymous codons for a particular amino acid, termed GCsyn—we try to deepen our understanding of the relationship between GC-content and amino acid/codon usage in more details. We analyzed 65 widely distributed representative species and found a close association between GCsyn, GC-content, and amino acids usage. The overall usages of the four amino acids with the greatest GCsyn and the five amino acids with the lowest GCsyn both vary with the regional GC-content, whereas the usage of the remaining 11 amino acids with intermediate GCsyn is less variable. More interesting, we discovered that codon usage frequencies are nearly constant in regions with similar GC-content. We further quantified the effects of regional GC-content variation (low to high) on amino acid usage and found that GC-content determines the usage variation of amino acids, especially those with extremely high GCsyn, which accounts for 76.7% of the changed GC-content for those regions. Our results suggest that GCsyn correlates with GC-content and has impact on codon/amino acid usage. These findings suggest a novel approach to understanding the role of codon and amino acid usage in shaping genomic architecture and evolutionary patterns of organisms. PMID:26248983

CCC CGA is a weak translational recoding site in Escherichia coli.

PubMed

Shu, Ping; Dai, Huacheng; Mandecki, Wlodek; Goldman, Emanuel

2004-12-08

Previously published experiments had indicated unexpected expression of a control vector in which a beta-galactosidase reporter was in the +1 reading frame relative to the translation start. This control vector contained the codon pair CCC CGA in the zero reading frame, raising the possibility that ribosomes rephased on this sequence, with peptidyl-tRNA(Pro) pairing with CCC in the +1 frame. This putative rephasing might also be exacerbated by the rare CGA Arg codon in the second position due to increased vacancy of the ribosomal A-site. To test this hypothesis, a series of site-directed mutants was constructed, including mutations in both the first and second codons of this codon pair. The results show that interrupting the continuous run of C residues with synonymous codon changes essentially abolishes the frameshift. Further, changing the rare Arg codon to a common Arg codon also reduces the frequency of the frameshift. These results provide strong support for the hypothesis that CCC CGA in the zero frame is indeed a weak translational frameshift site in Escherichia coli, with a 1-2% efficiency. Because the vector sequence also contains another CCC triplet in the +1 reading frame starting within the next codon after the CGA, our data also support possible contribution to expression of a +7 nucleotide ribosome hop into the same +1 reading frame. We also confirm here a previous report that CCC UGA is a translational frameshift site, in these experiments, with about 5% efficiency.

Glacial vicariance in Eurasia: mitochondrial DNA evidence from Scots pine for a complex heritage involving genetically distinct refugia at mid-northern latitudes and in Asia Minor

PubMed Central

Naydenov, Krassimir; Senneville, Sauphie; Beaulieu, Jean; Tremblay, Francine; Bousquet, Jean

2007-01-01

Background At the last glacial maximum, Fennoscandia was covered by an ice sheet while the tundra occupied most of the rest of northern Eurasia. More or less disjunct refugial populations of plants were dispersed in southern Europe, often trapped between mountain ranges and seas. Genetic and paleobotanical evidences indicate that these populations have contributed much to Holocene recolonization of more northern latitudes. Less supportive evidence has been found for the existence of glacial populations located closer to the ice margin. Scots pine (Pinus sylvestris L.) is a nordic conifer with a wide natural range covering much of Eurasia. Fractures in its extant genetic structure might be indicative of glacial vicariance and how different refugia contributed to the current distribution at the continental level. The population structure of Scots pine was investigated on much of its Eurasian natural range using maternally inherited mitochondrial DNA polymorphisms. Results A novel polymorphic region of the Scots pine mitochondrial genome has been identified, the intron 1 of nad7, with three variants caused by insertions-deletions. From 986 trees distributed among 54 populations, four distinct multi-locus mitochondrial haplotypes (mitotypes) were detected based on the three nad7 intron 1 haplotypes and two previously reported size variants for nad1 intron B/C. Population differentiation was high (GST = 0.657) and the distribution of the mitotypes was geographically highly structured, suggesting at least four genetically distinct ancestral lineages. A cosmopolitan lineage was widely distributed in much of Europe throughout eastern Asia. A previously reported lineage limited to the Iberian Peninsula was confirmed. A new geographically restricted lineage was found confined to Asia Minor. A new lineage was restricted to more northern latitudes in northeastern Europe and the Baltic region. Conclusion The contribution of the various ancestral lineages to the current distribution of Scots pine was asymmetric and extant endemism reflected the presence of large geographic barriers to migration. The results suggest a complex biogeographical history with glacial refugia shared with temperate plant species in southern European Peninsulas and Asia Minor, and a genetically distinct glacial population located more North. These results confirm recent observations for cold tolerant species about the possible existence of refugial populations at mid-northern latitudes contributing significantly to the recolonization of northern Europe. Thus, Eurasian populations of nordic plant species might not be as genetically homogenous as assumed by simply considering them as offsets of glacial populations located in southern peninsulas. As such, they might have evolved distinctive genetic adaptations during glacial vicariance, worth evaluating and considering for conservation. PMID:18034901

Sequence and structure-specific elements of HERG mRNA determine channel synthesis and trafficking efficiency

PubMed Central

Sroubek, Jakub; Krishnan, Yamini; McDonald, Thomas V.

2013-01-01

Human ether-á-gogo-related gene (HERG) encodes a potassium channel that is highly susceptible to deleterious mutations resulting in susceptibility to fatal cardiac arrhythmias. Most mutations adversely affect HERG channel assembly and trafficking. Why the channel is so vulnerable to missense mutations is not well understood. Since nothing is known of how mRNA structural elements factor in channel processing, we synthesized a codon-modified HERG cDNA (HERG-CM) where the codons were synonymously changed to reduce GC content, secondary structure, and rare codon usage. HERG-CM produced typical IKr-like currents; however, channel synthesis and processing were markedly different. Translation efficiency was reduced for HERG-CM, as determined by heterologous expression, in vitro translation, and polysomal profiling. Trafficking efficiency to the cell surface was greatly enhanced, as assayed by immunofluorescence, subcellular fractionation, and surface labeling. Chimeras of HERG-NT/CM indicated that trafficking efficiency was largely dependent on 5′ sequences, while translation efficiency involved multiple areas. These results suggest that HERG translation and trafficking rates are independently governed by noncoding information in various regions of the mRNA molecule. Noncoding information embedded within the mRNA may play a role in the pathogenesis of hereditary arrhythmia syndromes and could provide an avenue for targeted therapeutics.—Sroubek, J., Krishnan, Y., McDonald, T V. Sequence- and structure-specific elements of HERG mRNA determine channel synthesis and trafficking efficiency. PMID:23608144

Genome-wide analysis reveals class and gene specific codon usage adaptation in avian paramyxoviruses 1

USDA-ARS?s Scientific Manuscript database

In order to characterize the evolutionary adaptations of avian paramyxovirus 1 (APMV-1) genomes, we have compared codon usage and codon adaptation indexes among groups of Newcastle disease viruses that differ in biological, ecological, and genetic characteristics. We have used available GenBank com...

Enhanced expression of codon optimized Mycobacterium avium subsp. paratuberculosis antigens in Lactobacillus salivarius

USDA-ARS?s Scientific Manuscript database

We have previously identified the mycobacterial high G+C codon usage bias as a limiting factor in heterologous expression of MAP proteins from Lb.salivarius, and demonstrated that codon optimisation of a synthetic coding gene greatly enhances MAP protein production. Here, we effectively demonstrate ...

Complete mitochondrial genome of Palawan peacock-pheasant Polyplectron napoleonis (Galliformes, Phasianidae).

PubMed

Quach, Tommy; Brooks, Daniel M; Miranda, Hector C

2016-01-01

The complete mitochondrial genome of the Palawan peacock-pheasant Polyplectron napoleonis is 16,710 bp and contains 13 protein-coding genes, 2 rRNA genes, 22 tRNA genes and a control-region. All protein-coding genes use the standard ATG start codon, except for cox1 which has GTG start codon. Seven out of 13 PCGs have TAA stop codons, two have AGG (cox1 and nd6), and three PCGs (nd2, cox2 and nd4) have incomplete stop codon of just T- - nucleotide.

Codon Usage Bias and Determining Forces in Taenia solium Genome.

PubMed

Yang, Xing; Ma, Xusheng; Luo, Xuenong; Ling, Houjun; Zhang, Xichen; Cai, Xuepeng

2015-12-01

The tapeworm Taenia solium is an important human zoonotic parasite that causes great economic loss and also endangers public health. At present, an effective vaccine that will prevent infection and chemotherapy without any side effect remains to be developed. In this study, codon usage patterns in the T. solium genome were examined through 8,484 protein-coding genes. Neutrality analysis showed that T. solium had a narrow GC distribution, and a significant correlation was observed between GC12 and GC3. Examination of an NC (ENC vs GC3s)-plot showed a few genes on or close to the expected curve, but the majority of points with low-ENC (the effective number of codons) values were detected below the expected curve, suggesting that mutational bias plays a major role in shaping codon usage. The Parity Rule 2 plot (PR2) analysis showed that GC and AT were not used proportionally. We also identified 26 optimal codons in the T. solium genome, all of which ended with either a G or C residue. These optimal codons in the T. solium genome are likely consistent with tRNAs that are highly expressed in the cell, suggesting that mutational and translational selection forces are probably driving factors of codon usage bias in the T. solium genome.

Analyzing gene expression from relative codon usage bias in Yeast genome: a statistical significance and biological relevance.

PubMed

Das, Shibsankar; Roymondal, Uttam; Sahoo, Satyabrata

2009-08-15

Based on the hypothesis that highly expressed genes are often characterized by strong compositional bias in terms of codon usage, there are a number of measures currently in use that quantify codon usage bias in genes, and hence provide numerical indices to predict the expression levels of genes. With the recent advent of expression measure from the score of the relative codon usage bias (RCBS), we have explicitly tested the performance of this numerical measure to predict the gene expression level and illustrate this with an analysis of Yeast genomes. In contradiction with previous other studies, we observe a weak correlations between GC content and RCBS, but a selective pressure on the codon preferences in highly expressed genes. The assertion that the expression of a given gene depends on the score of relative codon usage bias (RCBS) is supported by the data. We further observe a strong correlation between RCBS and protein length indicating natural selection in favour of shorter genes to be expressed at higher level. We also attempt a statistical analysis to assess the strength of relative codon bias in genes as a guide to their likely expression level, suggesting a decrease of the informational entropy in the highly expressed genes.

Lost in Translation: Bioinformatic Analysis of Variations Affecting the Translation Initiation Codon in the Human Genome.

PubMed

Abad, Francisco; de la Morena-Barrio, María Eugenia; Fernández-Breis, Jesualdo Tomás; Corral, Javier

2018-06-01

Translation is a key biological process controlled in eukaryotes by the initiation AUG codon. Variations affecting this codon may have pathological consequences by disturbing the correct initiation of translation. Unfortunately, there is no systematic study describing these variations in the human genome. Moreover, we aimed to develop new tools for in silico prediction of the pathogenicity of gene variations affecting AUG codons, because to date, these gene defects have been wrongly classified as missense. Whole-exome analysis revealed the mean of 12 gene variations per person affecting initiation codons, mostly with high (> 0:01) minor allele frequency (MAF). Moreover, analysis of Ensembl data (December 2017) revealed 11,261 genetic variations affecting the initiation AUG codon of 7,205 genes. Most of these variations (99.5%) have low or unknown MAF, probably reflecting deleterious consequences. Only 62 variations had high MAF. Genetic variations with high MAF had closer alternative AUG downstream codons than did those with low MAF. Besides, the high-MAF group better maintained both the signal peptide and reading frame. These differentiating elements could help to determine the pathogenicity of this kind of variation. Data and scripts in Perl and R are freely available at https://github.com/fanavarro/hemodonacion. jfernand@um.es. Supplementary data are available at Bioinformatics online.

Influence of codon usage bias on FGLamide-allatostatin mRNA secondary structure.

PubMed

Martínez-Pérez, Francisco; Bendena, William G; Chang, Belinda S W; Tobe, Stephen S

2011-03-01

The FGLamide allatostatins (ASTs) are invertebrate neuropeptides which inhibit juvenile hormone biosynthesis in Dictyoptera and related orders. They also show myomodulatory activity. FGLamide AST nucleotide frequencies and codon bias were investigated with respect to possible effects on mRNA secondary structure. 367 putative FGLamide ASTs and their potential endoproteolytic cleavage sites were identified from 40 species of crustaceans, chelicerates and insects. Among these, 55% comprised only 11 amino acids. An FGLamide AST consensus was identified to be (X)(1→16)Y(S/A/N/G)FGLGKR, with a strong bias for the codons UUU encoding for Phe and AAA for Lys, which can form strong Watson-Crick pairing in all peptides analyzed. The physical distance between these codons favor a loop structure from Ser/Ala-Phe to Lys-Arg. Other loop and hairpin loops were also inferred from the codon frequencies in the N-terminal motif, and the first amino acids from the C-terminal motif, or the dibasic potential endoproteolytic cleavage site. Our results indicate that nucleotide frequencies and codon usage bias in FGLamide ASTs tend to favor mRNA folds in the codon sequence in the C-terminal active peptide core and at the dibasic potential endoproteolytic cleavage site. Copyright © 2010 Elsevier Inc. All rights reserved.

Influence of tree provenance on biogenic VOC emissions of Scots pine (Pinus sylvestris) stumps

NASA Astrophysics Data System (ADS)

Kivimäenpää, Minna; Magsarjav, Narantsetseg; Ghimire, Rajendra; Markkanen, Juha-Matti; Heijari, Juha; Vuorinen, Martti; Holopainen, Jarmo K.

2012-12-01

Resin-storing plant species such as conifer trees can release substantial amounts of volatile organic compounds (VOCs) into the atmosphere under stress circumstances that cause resin flow. Wounding can be induced by animals, pathogens, wind or direct mechanical damage e.g. during harvesting. In atmospheric modelling of biogenic VOCs, actively growing vegetation has been mostly considered as the source of emissions. Root systems and stumps of resin-storing conifer trees could constitute a significant store of resin after tree cutting. Therefore, we assessed the VOC emission rates from the cut surface of Scots pine stumps and estimated the average emission rates for an area with a density of 2000 stumps per ha. The experiment was conducted with trees of one Estonian and three Finnish Scots pine provenances covering a 1200 km gradient at a common garden established in central Finland in 1991. VOC emissions were dominated by monoterpenes and less than 0.1% of the total emission was sesquiterpenes. α-Pinene (7-92% of the total emissions) and 3-carene (0-76% of the total emissions) were the dominant monoterpenes. Proportions of α-pinene and camphene were significantly lower and proportions of 3-carene, sabinene, γ-terpinene and terpinolene higher in the southernmost Saaremaa provenance compared to the other provenances. Total terpene emission rates (standardised to +20 °C) from stumps varied from 27 to 1582 mg h-1 m-2 when measured within 2-3 h after tree cutting. Emission rates decreased rapidly to between 2 and 79 mg h-1 m-2 at 50 days after cutting. The estimated daily terpene emission rates on a hectare basis from freshly cut stumps at a cut tree density of 2000 per ha varied depending on provenance. Estimated emission ranges were 100-710 g ha-1 d-1 and 137-970 g ha-1 d-1 in 40 and in 60 year-old forest stands, respectively. Our result suggests that emission directly from stump surfaces could be a significant source of monoterpene emissions for a few weeks after logging in a Scots pine stand, but provenance properties strongly affect resin flow from root to stump surface.

Unproductively spliced ribosomal protein mRNAs are natural targets of mRNA surveillance in C. elegans

PubMed Central

Mitrovich, Quinn M.; Anderson, Philip

2000-01-01

Messenger RNA surveillance, the selective and rapid degradation of mRNAs containing premature stop codons, occurs in all eukaryotes tested. The biological role of this decay pathway, however, is not well understood. To identify natural substrates of mRNA surveillance, we used a cDNA-based representational difference analysis to identify mRNAs whose abundance increases in Caenorhabditis elegans smg(−) mutants, which are deficient for mRNA surveillance. Alternatively spliced mRNAs of genes encoding ribosomal proteins L3, L7a, L10a, and L12 are abundant natural targets of mRNA surveillance. Each of these genes expresses two distinct mRNAs. A productively spliced mRNA, whose abundance does not change in smg(−) mutants, encodes a normal, full-length, ribosomal protein. An unproductively spliced mRNA, whose abundance increases dramatically in smg(−) mutants, contains premature stop codons because of incomplete removal of an alternatively spliced intron. In transgenic animals expressing elevated quantities of RPL-12, a greater proportion of endogenous rpl-12 transcript is spliced unproductively. Thus, RPL-12 appears to autoregulate its own splicing, with unproductively spliced mRNAs being degraded by mRNA surveillance. We demonstrate further that alternative splicing of rpl introns is conserved among widely diverged nematodes. Our results suggest that one important role of mRNA surveillance is to eliminate unproductive by-products of gene regulation. PMID:10970881

Determinants of translation speed are randomly distributed across transcripts resulting in a universal scaling of protein synthesis times

NASA Astrophysics Data System (ADS)

Sharma, Ajeet K.; Ahmed, Nabeel; O'Brien, Edward P.

2018-02-01

Ribosome profiling experiments have found greater than 100-fold variation in ribosome density along mRNA transcripts, indicating that individual codon elongation rates can vary to a similar degree. This wide range of elongation times, coupled with differences in codon usage between transcripts, suggests that the average codon translation-rate per gene can vary widely. Yet, ribosome run-off experiments have found that the average codon translation rate for different groups of transcripts in mouse stem cells is constant at 5.6 AA/s. How these seemingly contradictory results can be reconciled is the focus of this study. Here, we combine knowledge of the molecular factors shown to influence translation speed with genomic information from Escherichia coli, Saccharomyces cerevisiae and Homo sapiens to simulate the synthesis of cytosolic proteins in these organisms. The model recapitulates a near constant average translation rate, which we demonstrate arises because the molecular determinants of translation speed are distributed nearly randomly amongst most of the transcripts. Consequently, codon translation rates are also randomly distributed and fast-translating segments of a transcript are likely to be offset by equally probable slow-translating segments, resulting in similar average elongation rates for most transcripts. We also show that the codon usage bias does not significantly affect the near random distribution of codon translation rates because only about 10 % of the total transcripts in an organism have high codon usage bias while the rest have little to no bias. Analysis of Ribo-Seq data and an in vivo fluorescent assay supports these conclusions.

Idiosyncratic recognition of UUG/UUA codons by modified nucleoside 5-taurinomethyluridine, τm5U present at 'wobble' position in anticodon loop of tRNALeu: A molecular modeling approach.

PubMed

Kamble, Asmita S; Fandilolu, Prayagraj M; Sambhare, Susmit B; Sonawane, Kailas D

2017-01-01

Lack of naturally occurring modified nucleoside 5-taurinomethyluridine (τm5U) at the 'wobble' 34th position in tRNALeu causes mitochondrial myopathy, encephalopathy, lactic acidosis and stroke-like episodes (MELAS). The τm5U34 specifically recognizes UUG and UUA codons. Structural consequences of τm5U34 to read cognate codons have not been studied so far in detail at the atomic level. Hence, 50ns multiple molecular dynamics (MD) simulations of various anticodon stem loop (ASL) models of tRNALeu in presence and absence of τm5U34 along with UUG and UUA codons were performed to explore the dynamic behaviour of τm5U34 during codon recognition process. The MD simulation results revealed that τm5U34 recognizes G/A ending codons by 'wobble' as well as a novel 'single' hydrogen bonding interactions. RMSD and RMSF values indicate the comparative stability of the ASL models containing τm5U34 modification over the other models, lacking τm5U34. Another MD simulation study of 55S mammalian mitochondrial rRNA with tRNALeu showed crucial interactions between the A-site residues, A918, A919, G256 and codon-anticodon bases. Thus, these results could improve our understanding about the decoding efficiency of human mt tRNALeu with τm5U34 to recognize UUG and UUA codons.

Idiosyncratic recognition of UUG/UUA codons by modified nucleoside 5-taurinomethyluridine, τm5U present at ‘wobble’ position in anticodon loop of tRNALeu: A molecular modeling approach

PubMed Central

Kamble, Asmita S.; Fandilolu, Prayagraj M.; Sambhare, Susmit B.; Sonawane, Kailas D.

2017-01-01

Lack of naturally occurring modified nucleoside 5-taurinomethyluridine (τm5U) at the ‘wobble’ 34th position in tRNALeu causes mitochondrial myopathy, encephalopathy, lactic acidosis and stroke-like episodes (MELAS). The τm5U34 specifically recognizes UUG and UUA codons. Structural consequences of τm5U34 to read cognate codons have not been studied so far in detail at the atomic level. Hence, 50ns multiple molecular dynamics (MD) simulations of various anticodon stem loop (ASL) models of tRNALeu in presence and absence of τm5U34 along with UUG and UUA codons were performed to explore the dynamic behaviour of τm5U34 during codon recognition process. The MD simulation results revealed that τm5U34 recognizes G/A ending codons by ‘wobble’ as well as a novel ‘single’ hydrogen bonding interactions. RMSD and RMSF values indicate the comparative stability of the ASL models containing τm5U34 modification over the other models, lacking τm5U34. Another MD simulation study of 55S mammalian mitochondrial rRNA with tRNALeu showed crucial interactions between the A-site residues, A918, A919, G256 and codon-anticodon bases. Thus, these results could improve our understanding about the decoding efficiency of human mt tRNALeu with τm5U34 to recognize UUG and UUA codons. PMID:28453549

Recent evidence for evolution of the genetic code

NASA Technical Reports Server (NTRS)

Osawa, S.; Jukes, T. H.; Watanabe, K.; Muto, A.

1992-01-01

The genetic code, formerly thought to be frozen, is now known to be in a state of evolution. This was first shown in 1979 by Barrell et al. (G. Barrell, A. T. Bankier, and J. Drouin, Nature [London] 282:189-194, 1979), who found that the universal codons AUA (isoleucine) and UGA (stop) coded for methionine and tryptophan, respectively, in human mitochondria. Subsequent studies have shown that UGA codes for tryptophan in Mycoplasma spp. and in all nonplant mitochondria that have been examined. Universal stop codons UAA and UAG code for glutamine in ciliated protozoa (except Euplotes octacarinatus) and in a green alga, Acetabularia. E. octacarinatus uses UAA for stop and UGA for cysteine. Candida species, which are yeasts, use CUG (leucine) for serine. Other departures from the universal code, all in nonplant mitochondria, are CUN (leucine) for threonine (in yeasts), AAA (lysine) for asparagine (in platyhelminths and echinoderms), UAA (stop) for tyrosine (in planaria), and AGR (arginine) for serine (in several animal orders) and for stop (in vertebrates). We propose that the changes are typically preceded by loss of a codon from all coding sequences in an organism or organelle, often as a result of directional mutation pressure, accompanied by loss of the tRNA that translates the codon. The codon reappears later by conversion of another codon and emergence of a tRNA that translates the reappeared codon with a different assignment. Changes in release factors also contribute to these revised assignments. We also discuss the use of UGA (stop) as a selenocysteine codon and the early history of the code.

A condition-specific codon optimization approach for improved heterologous gene expression in Saccharomyces cerevisiae

PubMed Central

2014-01-01

Background Heterologous gene expression is an important tool for synthetic biology that enables metabolic engineering and the production of non-natural biologics in a variety of host organisms. The translational efficiency of heterologous genes can often be improved by optimizing synonymous codon usage to better match the host organism. However, traditional approaches for optimization neglect to take into account many factors known to influence synonymous codon distributions. Results Here we define an alternative approach for codon optimization that utilizes systems level information and codon context for the condition under which heterologous genes are being expressed. Furthermore, we utilize a probabilistic algorithm to generate multiple variants of a given gene. We demonstrate improved translational efficiency using this condition-specific codon optimization approach with two heterologous genes, the fluorescent protein-encoding eGFP and the catechol 1,2-dioxygenase gene CatA, expressed in S. cerevisiae. For the latter case, optimization for stationary phase production resulted in nearly 2.9-fold improvements over commercial gene optimization algorithms. Conclusions Codon optimization is now often a standard tool for protein expression, and while a variety of tools and approaches have been developed, they do not guarantee improved performance for all hosts of applications. Here, we suggest an alternative method for condition-specific codon optimization and demonstrate its utility in Saccharomyces cerevisiae as a proof of concept. However, this technique should be applicable to any organism for which gene expression data can be generated and is thus of potential interest for a variety of applications in metabolic and cellular engineering. PMID:24636000

Alterations of the three short open reading frames in the Rous sarcoma virus leader RNA modulate viral replication and gene expression.

PubMed Central

Moustakas, A; Sonstegard, T S; Hackett, P B

1993-01-01

The Rous sarcoma virus (RSV) leader RNA has three short open reading frames (ORF1 to ORF3) which are conserved in all avian sarcoma-leukosis retroviruses. Effects on virus propagation were determined following three types of alterations in the ORFs: (i) replacement of AUG initiation codons in order to prohibit ORF translation, (ii) alterations of the codon context around the AUG initiation codon to enhance translation of the normally silent ORF3, and (iii) elongation of the ORF coding sequences. Mutagenesis of the AUG codons for ORF1 and ORF2 (AUG1 and AUG2) singly or together delayed the onset of viral replication and cell transformation. In contrast, mutagenesis of AUG3 almost completely suppressed these viral activities. Mutagenesis of ORF3 to enhance its translation inhibited viral propagation. When the mutant ORF3 included an additional frameshift mutation which extended the ORF beyond the initiation site for the gag, gag-pol, and env proteins, host cells were initially transformed but died soon thereafter. Elongation of ORF1 from 7 to 62 codons led to the accumulation of transformation-defective virus with a delayed onset of replication. In contrast, viruses with elongation of ORF1 from 7 to 30 codons, ORF2 from 16 to 48 codons, or ORF3 from 9 to 64 codons, without any alterations in the AUG context, exhibited wild-type phenotypes. These results are consistent with a model that translation of the ORFs is necessary to facilitate virus production. Images PMID:7685415

Codon usage bias reveals genomic adaptations to environmental conditions in an acidophilic consortium.

PubMed

Hart, Andrew; Cortés, María Paz; Latorre, Mauricio; Martinez, Servet

2018-01-01

The analysis of codon usage bias has been widely used to characterize different communities of microorganisms. In this context, the aim of this work was to study the codon usage bias in a natural consortium of five acidophilic bacteria used for biomining. The codon usage bias of the consortium was contrasted with genes from an alternative collection of acidophilic reference strains and metagenome samples. Results indicate that acidophilic bacteria preferentially have low codon usage bias, consistent with both their capacity to live in a wide range of habitats and their slow growth rate, a characteristic probably acquired independently from their phylogenetic relationships. In addition, the analysis showed significant differences in the unique sets of genes from the autotrophic species of the consortium in relation to other acidophilic organisms, principally in genes which code for proteins involved in metal and oxidative stress resistance. The lower values of codon usage bias obtained in this unique set of genes suggest higher transcriptional adaptation to living in extreme conditions, which was probably acquired as a measure for resisting the elevated metal conditions present in the mine.

RNA editing makes mistakes in plant mitochondria: editing loses sense in transcripts of a rps19 pseudogene and in creating stop codons in coxI and rps3 mRNAs of Oenothera.

PubMed Central

Schuster, W; Brennicke, A

1991-01-01

An intact gene for the ribosomal protein S19 (rps19) is absent from Oenothera mitochondria. The conserved rps19 reading frame found in the mitochondrial genome is interrupted by a termination codon. This rps19 pseudogene is cotranscribed with the downstream rps3 gene and is edited on both sides of the translational stop. Editing, however, changes the amino acid sequence at positions that were well conserved before editing. Other strange editings create translational stops in open reading frames coding for functional proteins. In coxI and rps3 mRNAs CGA codons are edited to UGA stop codons only five and three codons, respectively, downstream to the initiation codon. These aberrant editings in essential open reading frames and in the rps19 pseudogene appear to have been shifted to these positions from other editing sites. These observations suggest a requirement for a continuous evolutionary constraint on the editing specificities in plant mitochondria. Images PMID:1762921

Identification of four novel HLA-B alleles, B*1590, B*1591, B*2726, and B*4705, from an East African population by high-resolution sequence-based typing.

PubMed

Luo, M; Mao, X; Plummer, F A

2005-02-01

We report here four novel HLA-B alleles, B*1590, B*1591, B*2726, and B*4705, identified from an East African population during sequence-based HLA-B typing. The novel alleles were confirmed by sequencing two separate polymerase chain reaction products, and by molecular cloning and sequencing multiple clones. B*1590 is identical to B*1510 at exon 2 and exon 3, except for a difference (GCCGTC) at codon 158. Sequence differences at codon 152 (GAGGTG) and codon 167 (TGGTCG) differentiate B*1591 from B*1503 at exon 3. B*2726 is identical to B*2708 at exon 2 and exon 3, except for a difference (AAGCAG) at codon 70. B*4705 was identified in three Kenyan women. The allele is identical to B*47010101/02 at exon 2 and exon 3, except for differences at codon 97 (AGGAAT) and codon 99 (TTTTAT). These new alleles have been named by the WHO Nomenclature Committee. Identification of these novel HLA-B alleles reflects the genetic diversity of this East African population.

Energetics of codon-anticodon recognition on the small ribosomal subunit.

PubMed

Almlöf, Martin; Andér, Martin; Aqvist, Johan

2007-01-09

Recent crystal structures of the small ribosomal subunit have made it possible to examine the detailed energetics of codon recognition on the ribosome by computational methods. The binding of cognate and near-cognate anticodon stem loops to the ribosome decoding center, with mRNA containing the Phe UUU and UUC codons, are analyzed here using explicit solvent molecular dynamics simulations together with the linear interaction energy (LIE) method. The calculated binding free energies are in excellent agreement with experimental binding constants and reproduce the relative effects of mismatches in the first and second codon position versus a mismatch at the wobble position. The simulations further predict that the Leu2 anticodon stem loop is about 10 times more stable than the Ser stem loop in complex with the Phe UUU codon. It is also found that the ribosome significantly enhances the intrinsic stability differences of codon-anticodon complexes in aqueous solution. Structural analysis of the simulations confirms the previously suggested importance of the universally conserved nucleotides A1492, A1493, and G530 in the decoding process.

Simple-MSSM: a simple and efficient method for simultaneous multi-site saturation mutagenesis.

PubMed

Cheng, Feng; Xu, Jian-Miao; Xiang, Chao; Liu, Zhi-Qiang; Zhao, Li-Qing; Zheng, Yu-Guo

2017-04-01

To develop a practically simple and robust multi-site saturation mutagenesis (MSSM) method that enables simultaneously recombination of amino acid positions for focused mutant library generation. A general restriction enzyme-free and ligase-free MSSM method (Simple-MSSM) based on prolonged overlap extension PCR (POE-PCR) and Simple Cloning techniques. As a proof of principle of Simple-MSSM, the gene of eGFP (enhanced green fluorescent protein) was used as a template gene for simultaneous mutagenesis of five codons. Forty-eight randomly selected clones were sequenced. Sequencing revealed that all the 48 clones showed at least one mutant codon (mutation efficiency = 100%), and 46 out of the 48 clones had mutations at all the five codons. The obtained diversities at these five codons are 27, 24, 26, 26 and 22, respectively, which correspond to 84, 75, 81, 81, 69% of the theoretical diversity offered by NNK-degeneration (32 codons; NNK, K = T or G). The enzyme-free Simple-MSSM method can simultaneously and efficiently saturate five codons within one day, and therefore avoid missing interactions between residues in interacting amino acid networks.

Lack of correlation between p53 codon 72 polymorphism and anal cancer risk

PubMed Central

Contu, Simone S; Agnes, Grasiela; Damin, Andrea P; Contu, Paulo C; Rosito, Mário A; Alexandre, Claudio O; Damin, Daniel C

2009-01-01

AIM: To investigate the potential role of p53 codon 72 polymorphism as a risk factor for development of anal cancer. METHODS: Thirty-two patients with invasive anal carcinoma and 103 healthy blood donors were included in the study. p53 codon 72 polymorphism was analyzed in blood samples through polymerase chain reaction-restriction fragment length polymorphism and DNA sequencing. RESULTS: The relative frequency of each allele was 0.60 for Arg and 0.40 for Pro in patients with anal cancer, and 0.61 for Arg and 0.39 for Pro in normal controls. No significant differences in distribution of the codon 72 genotypes between patients and controls were found. CONCLUSION: These results do not support a role for the p53 codon 72 polymorphism in anal carcinogenesis. PMID:19777616

Molecular Scanning of β-Thalassemia in the Southern Region of Central Java, Indonesia; a Step Towards a Local Prevention Program.

PubMed

Rujito, Lantip; Basalamah, Muhammad; Mulatsih, Sri; Sofro, Abdul Salam M

2015-08-03

Thalassemia is the most prevalent genetic blood disorder worldwide, and particularly prevalent in Indonesia. The purpose of this study was to determine the spectrum of β-thalassemia (β-thal) mutations found in the southern region of Central Java, Indonesia. The subjects of the study included 209 β-thal Javanese patients from Banyumas Residency, a southwest region of Central Java Province. DNA analysis was performed using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP), amplification refractory mutation system (ARMS), and the direct sequencing method. The results showed that 14 alleles were found in the following order: IVS-I-5 (G > C) (HBB: c.92 + 5G > C) 43.5%, codon 26 (Hb E; HBB: c.79G > A) 28.2%, IVS-I-1 (G > A) (HBB: c.92 + 1G > A) 5.0%, codon 15 (TGG > TAG) (HBB: c.47G > A) 3.8%, IVS-I-1 (G > T) (HBB: c.92 + 1G > T) 3.1%, codon 35 (-C) (HBB: c.110delC) 2.4%. The rest, including codons 41/42 (-TTCT) (HBB: c.126_129delCTTT), codons 8/9 (+G) (HBB: c.27_28insG), codon 19 (AAC > AGC) (HBB: c.59A > G), codon 17 (AAG > TAG) (HBB: c.52A > T), IVS-I-2 (T > C) (HBB: c.92 + 2T > C), codons 123/124/125 (-ACCCCACC) (HBB: c.370_378delACCCCACCA), codon 40 (-G) (HBB: c.123delG) and Cap +1 (A > C) (HBB: c.-50A > C), accounted for up to 1.0% each. The most prevalent alleles would be recommended to be used as part of β-thal screening for the Javanese, one of the major ethnic groups in the country.

Random codon re-encoding induces stable reduction of replicative fitness of Chikungunya virus in primate and mosquito cells.

PubMed

Nougairede, Antoine; De Fabritus, Lauriane; Aubry, Fabien; Gould, Ernest A; Holmes, Edward C; de Lamballerie, Xavier

2013-02-01

Large-scale codon re-encoding represents a powerful method of attenuating viruses to generate safe and cost-effective vaccines. In contrast to specific approaches of codon re-encoding which modify genome-scale properties, we evaluated the effects of random codon re-encoding on the re-emerging human pathogen Chikungunya virus (CHIKV), and assessed the stability of the resultant viruses during serial in cellulo passage. Using different combinations of three 1.4 kb randomly re-encoded regions located throughout the CHIKV genome six codon re-encoded viruses were obtained. Introducing a large number of slightly deleterious synonymous mutations reduced the replicative fitness of CHIKV in both primate and arthropod cells, demonstrating the impact of synonymous mutations on fitness. Decrease of replicative fitness correlated with the extent of re-encoding, an observation that may assist in the modulation of viral attenuation. The wild-type and two re-encoded viruses were passaged 50 times either in primate or insect cells, or in each cell line alternately. These viruses were analyzed using detailed fitness assays, complete genome sequences and the analysis of intra-population genetic diversity. The response to codon re-encoding and adaptation to culture conditions occurred simultaneously, resulting in significant replicative fitness increases for both re-encoded and wild type viruses. Importantly, however, the most re-encoded virus failed to recover its replicative fitness. Evolution of these viruses in response to codon re-encoding was largely characterized by the emergence of both synonymous and non-synonymous mutations, sometimes located in genomic regions other than those involving re-encoding, and multiple convergent and compensatory mutations. However, there was a striking absence of codon reversion (<0.4%). Finally, multiple mutations were rapidly fixed in primate cells, whereas mosquito cells acted as a brake on evolution. In conclusion, random codon re-encoding provides important information on the evolution and genetic stability of CHIKV viruses and could be exploited to develop a safe, live attenuated CHIKV vaccine.

Molecular Scanning of β-Thalassemia in the Southern Region of Central Java, Indonesia; a Step Towards a Local Prevention Program.

PubMed

Rujito, Lantip; Basalamah, Muhammad; Mulatsih, Sri; Sofro, Abdul Salam M

2015-01-01

Thalassemia is the most prevalent genetic blood disorder worldwide, and particularly prevalent in Indonesia. The purpose of this study was to determine the spectrum of β-thalassemia (β-thal) mutations found in the southern region of Central Java, Indonesia. The subjects of the study included 209 β-thal Javanese patients from Banyumas Residency, a southwest region of Central Java Province. DNA analysis was performed using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP), amplification refractory mutation system (ARMS), and the direct sequencing method. The results showed that 14 alleles were found in the following order: IVS-I-5 (G > C) (HBB: c.92 + 5G > C) 43.5%, codon 26 (Hb E; HBB: c.79G > A) 28.2%, IVS-I-1 (G > A) (HBB: c.92 + 1G > A) 5.0%, codon 15 (TGG > TAG) (HBB: c.47G > A) 3.8%, IVS-I-1 (G > T) (HBB: c.92 + 1G > T) 3.1%, codon 35 (-C) (HBB: c.110delC) 2.4%. The rest, including codons 41/42 (-TTCT) (HBB: c.126_129delCTTT), codons 8/9 (+G) (HBB: c.27_28insG), codon 19 (AAC > AGC) (HBB: c.59A > G), codon 17 (AAG > TAG) (HBB: c.52A > T), IVS-I-2 (T > C) (HBB: c.92 + 2T > C), codons 123/124/125 (-ACCCCACC) (HBB: c.370_378delACCCCACCA), codon 40 (-G) (HBB: c.123delG) and Cap +1 (A > C) (HBB: c.-50A > C), accounted for up to 1.0% each. The most prevalent alleles would be recommended to be used as part of β-thal screening for the Javanese, one of the major ethnic groups in the country.

Selectivity of Pinus sylvestris extract and essential oil to estrogen-insensitive breast cancer cells Pinus sylvestris against cancer cells.

PubMed

Hoai, Nguyen Thi; Duc, Ho Viet; Thao, Do Thi; Orav, Anne; Raal, Ain

2015-10-01

So far, the anticancer action of pine tree extracts has mainly been shown for the species distributed widely around the Asian countries. Therefore, this study was performed to examine the potential cytotoxicity of Scots pine (Pinus sylvestris L.) native also to the European region and growing widely in Estonia. The cytotoxic activity of methanol extract and essential oil of Scots pine needles was determined by sulforhodamine B assay in different human cancer cell lines. This needle extract was found to suppress the viability of several human cancer cell lines showing some selectivity to estrogen receptor negative breast cancer cells, MDA-MB-231(half maximal inhibitory concentration [IC50] 35 μg/ml) in comparison with estrogen receptor-positive breast cancer cells, MCF-7 (IC50 86 μg/ml). It is the strongest cytotoxic effect at all measured, thus far for the needles and leaves extracts derived from various pine species, and is also the first study comparing the anticancer effects of pine tree extracts on molecularly different human breast cancer cells. The essential oil showed the stronger cytotoxic effect to both negative and positive breast cancer cell lines (both IC50 29 μg/ml) than pine extract (IC50 42 and 80 μg/ml, respectively). The data from this report indicate that Scots pine needles extract and essential oil exhibits some potential as chemopreventive or chemotherapeutic agent for mammary tumors unresponsive to endocrine treatment.

Effects of male fecundity, interindividual distance and anisotropic pollen dispersal on mating success in a Scots pine (Pinus sylvestris) seed orchard

PubMed Central

Torimaru, T; Wennström, U; Lindgren, D; Wang, X-R

2012-01-01

Quantifying the effect of pollen dispersal and flowering traits on mating success is essential for understanding evolutionary responses to changing environments and establishing strategies for forest tree breeding. This study examined, quantitatively, the effects of male fecundity, interindividual distance and anisotropic pollen dispersal on the mating success of Scots pine (Pinus sylvestris), utilizing a well-mapped Scots pine seed orchard. Paternity analysis of 1021 seeds sampled from 87 trees representing 28 clones showed that 53% of the seeds had at least one potential pollen parent within the orchard. Pronounced variation in paternal contribution was observed among clones. Variations in pollen production explained up to 78% of the variation in mating success, which was 11.2 times greater for clones producing the largest amount of pollen than for clones producing the least pollen. Mating success also varied with intertree distance and direction, which explained up to 28% of the variance. Fertilization between neighboring trees 2.3 m apart was 2.4 times more frequent than between trees 4.6 m apart, and up to 12.4 times higher for trees downwind of the presumed prevailing wind direction than for upwind trees. The effective number of pollen donors recorded in the seed orchard (12.2) was smaller than the theoretical expectation (19.7). Based on the empirical observations, a mating model that best describes the gene dispersal pattern in clonal seed orchards was constructed. PMID:21897440

Association of FLOWERING LOCUS T/TERMINAL FLOWER 1-like gene FTL2 expression with growth rhythm in Scots pine (Pinus sylvestris).

PubMed

Avia, Komlan; Kärkkäinen, Katri; Lagercrantz, Ulf; Savolainen, Outi

2014-10-01

Understanding the genetic basis of the timing of bud set, an important trait in conifers, is relevant for adaptation and forestry practice. In common garden experiments, both Scots pine (Pinus sylvestris) and Norway spruce (Picea abies) show a latitudinal cline in the trait. We compared the regulation of their bud set biology by examining the expression of PsFTL2, a Pinus sylvestris homolog to PaFTL2, a FLOWERING LOCUS T/TERMINAL FLOWER 1 (FT/TFL1)-like gene, the expression levels of which have been found previously to be associated with the timing of bud set in Norway spruce. In a common garden study, we analyzed the relationship of bud phenology under natural and artificial photoperiods and the expression of PsFTL2 in a set of Scots pine populations from different latitudes. The expression of PsFTL2 increased in the needles preceding bud set and decreased during bud burst. In the northernmost population, even short night periods were efficient to trigger this expression, which also increased earlier under all photoperiodic regimes compared with the southern populations. Despite the different biology, with few limitations, the two conifers that diverged 140 million yr ago probably share an association of FTL2 with bud set, pointing to a common mechanism for the timing of growth cessation in conifers. © 2014 The Authors. New Phytologist © 2014 New Phytologist Trust.

No evidence for depletion of carbohydrate pools in Scots pine (Pinus sylvestris L.) under drought stress

PubMed Central

Gruber, A.; Pirkebner, D.; Florian, C.; Oberhuber, W.

2012-01-01

The physiological mechanisms leading to Scots pine (Pinus sylvestris L.) decline in the dry inner Alpine valleys are still unknown. Testing the carbon starvation hypothesis, we analysed the seasonal course of mobile carbohydrate pools (NSC) of Scots pine growing at a xeric and a dry-mesic site within an inner Alpine dry valley (750 m a.s.l., Tyrol, Austria) during the year 2009, which was characterized by exceptional soil dryness. Although, soil moisture content dropped to c. 10% at both sites during the growing season, NSC concentrations were rising in all tissues (branch, stem, root) till end of July, except in needles where maxima were reached around bud break. NSC concentrations were not significantly different in the analysed tissues at the xeric and the dry-mesic site. At the dry-mesic site NSC concentrations in the above ground tree biomass were significantly higher during the period of radial growth. An accumulation of NSC in roots at the end of July indicates a change in carbon allocation after an early cessation in above ground growth, possibly due to elevated below ground carbon demand. In conclusion our results revealed that extensive soil dryness during the growing season did not lead to carbon depletion. However, even though C-reserves were not exhausted, a sequestration of carbohydrate pools during drought periods might lead to deficits in carbon supply that weaken tree vigour and drive tree mortality. PMID:21974742

Assessment of growth and stemwood quality of Scots pine on territory influenced by alkaline industrial dust.

PubMed

Mandre, Malle; Kask, Regino; Pikk, Jaak; Ots, Katri

2008-03-01

Long-term influence of alkaline dust (pH 12.3-12.7) pollution emitted over 40 years from a cement plant in Estonia was the reason of alkalisation (pH 6.7-7.9) and high concentrations of K, Ca and Mg in the soil of affected territories. Although dust emission has diminished during the last 10 years, the imbalances in nutrition substrate and their influence on the growth of trees are notable up to now. The study of morphological and physical properties of 70-80-year-old Scots pine (Pinus sylvestris L.) crown, stems and stemwood from three different air pollution zones showed serious deviations in comparison with a relatively healthy forest in an unpolluted area. The specimens from polluted trees, if compared to reference site, showed significantly smaller height growth, radial increment and width of annual rings of sapwood. In heartwood wider annual rings were found in polluted areas. In the period of heartwood formation the dust pollution level emitted from the plant was relatively modest and cement dust, which contains elements necessary for mineral nutrition of trees, may have acted as fertiliser. The moisture content in sapwood and heartwood, especially in the upper layers of stems, was lower in the polluted area than in reference site trees. Regression analysis revealed a strong dependence between latewood percentage and sapwood or heartwood in stems of Scots pine in all sample plots.

Does tree diversity increase wood production in pine forests?

PubMed

Vilà, Montserrat; Vayreda, Jordi; Gracia, Carles; Ibáñez, Joan Josep

2003-04-01

Recent experimental advances on the positive effect of species richness on ecosystem productivity highlight the need to explore this relationship in communities other than grasslands and using non-synthetic experiments. We investigated whether wood production in forests dominated by Aleppo pine (Pinus halepensis) and Pyrenean Scots pine (Pinus sylvestris) differed between monospecific and mixed forests (2-5 species) using the Ecological and Forest Inventory of Catalonia (IEFC) database which contains biotic and environmental characteristics for 10,644 field plots distributed within a 31,944 km(2) area in Catalonia (NE Spain). We found that in Pyrenean Scots pine forests wood production was not significantly different between monospecific and mixed plots. In contrast, in Aleppo pine forests wood production was greater in mixed plots than in monospecific plots. However, when climate, bedrock types, radiation and successional stage per plot were included in the analysis, species richness was no longer a significant factor. Aleppo pine forests had the highest productivity in plots located in humid climates and on marls and sandstone bedrocks. Climate did not influence wood production in Pyrenean Scots pine forests, but it was highest on sandstone and consolidated alluvial materials. For both pine forests wood production was negatively correlated with successional stage. Radiation did not influence wood production. Our analysis emphasizes the influence of macroenvironmental factors and temporal variation on tree productivity at the regional scale. Well-conducted forest surveys are an excellent source of data to test for the association between diversity and productivity driven by large-scale environmental factors.

Genetic and serological diversity of Flavobacterium psychrophilum isolates from salmonids in United Kingdom.

PubMed

Ngo, Thao P H; Bartie, Kerry L; Thompson, Kim D; Verner-Jeffreys, David W; Hoare, Rowena; Adams, Alexandra

2017-03-01

Flavobacterium psychrophilum is one of the most important bacterial pathogens affecting cultured rainbow trout (Oncorhynchus mykiss) and is increasingly causing problems in Atlantic salmon (Salmo salar L.) hatcheries. Little is known about the heterogeneity of F. psychrophilum isolates on UK salmonid farms. A total of 315 F. psychrophilum isolates, 293 of which were collected from 27 sites within the UK, were characterised using four genotyping methods and a serotyping scheme. A high strain diversity was identified among the isolates with 54 pulsotypes, ten (GTG) 5 -PCR types, two 16S rRNA allele lineages, seven plasmid profiles and three serotypes. Seven PFGE groups and 27 singletons were formed at a band similarity of 80%. PFGE group P (n=75) was found to be numerically predominant in eight sites within the UK. Two major PFGE clusters and 13 outliers were found at the band similarity of 40%. The predominant profileobserved within the F. psychrophilum isolates examined was PFGE cluster II - (GTG) 5 -PCR type r1-16S rRNA lineage II - serotype Th (70/156 isolates examined, 45%). Co-existence of genetically and serologically heterogeneous isolates within each farm was detected, confounding the ability to control RTFS outbreaks. The occurrence over time (up to 11 years) of F. psychrophilum pulsotypes in three representative sites (Scot I, Scot III and Scot V) within Scotland was examined, potentially providing important epidemiological data for farm management and the development of site-specific vaccines. Copyright © 2017 Elsevier B.V. All rights reserved.

Radial diffusion, vertical transport, and refixation of labeled bicarbonate in scots pine stems

NASA Astrophysics Data System (ADS)

Marshall, J. D.; Tarvainen, L.; Wallin, G.

2016-12-01

The CO2 produced by a respiring stem provides an index of metabolic activity in the stem and a quantitative estimate of an important component of the forest carbon budget. Production of CO2 by a given stem volume is lost by three competing processes. First, some diffuses radially outward through the bark. Second, some is dissolved and vertically transported upward out of the control volume by the xylem stream. Third, some is refixed by photosynthesis under the bark. The relative balance among these pathways was quantified in 17-m Scots pine trees by 13C-bicarbonate labeling of the xylem stream and monitoring of the 13CO2 in the xylem water, along with continuous monitoring of the radial diffusive flux at four canopy heights and in transpiration from leaves. Most of the label diffused out radially, as 13CO2, immediately above the labeling site, over about a week. The pulse was weakly and briefly detected 4 m above that height. Further up the stem it was not detected at all. We detected significant refixation of CO2 in the stems at all heights above 4 m, where the bark becomes papery and thin, but the label was so weak at this height that refixation had little influence on the pulse chase. We conclude that the vertical flux is negligible in Scots pine, but that the refixation flux must be accounted for in estimates of whole-stem CO2 efflux.

No evidence for depletion of carbohydrate pools in Scots pine (Pinus sylvestris L.) under drought stress.

PubMed

Gruber, A; Pirkebner, D; Florian, C; Oberhuber, W

2012-01-01

The physiological mechanisms leading to Scots pine (Pinus sylvestris L.) decline in the dry inner alpine valleys are still unknown. Testing the carbon starvation hypothesis, we analysed the seasonal course of mobile carbohydrate pools (NSC) of Scots pine growing at a xeric and a dry-mesic site within an inner alpine dry valley (750 m a.s.l., Tyrol, Austria) during 2009, which was characterised by exceptional soil dryness. Although, soil moisture content dropped to ca. 10% at both sites during the growing season, NSC concentrations rose in all tissues (branch, stem, root) until the end of July, except in needles, where maxima were reached around bud break. NSC concentrations were not significantly different in the analysed tissues at the xeric and the dry-mesic site. At the dry-mesic site, NSC concentrations in the aboveground tree biomass were significantly higher during the period of radial growth. An accumulation of NSC in roots at the end of July indicates a change in carbon allocation after an early cessation in aboveground growth, possibly due to elevated belowground carbon demand. In conclusion, our results revealed that extensive soil dryness during the growing season did not lead to carbon depletion. However, even though carbon reserves were not exhausted, sequestration of carbohydrate pools during drought periods might lead to deficits in carbon supply that weaken tree vigour and drive tree mortality. © 2011 German Botanical Society and The Royal Botanical Society of the Netherlands.

Large-scale analyses of synonymous substitution rates can be sensitive to assumptions about the process of mutation.

PubMed

Aris-Brosou, Stéphane; Bielawski, Joseph P

2006-08-15

A popular approach to examine the roles of mutation and selection in the evolution of genomes has been to consider the relationship between codon bias and synonymous rates of molecular evolution. A significant relationship between these two quantities is taken to indicate the action of weak selection on substitutions among synonymous codons. The neutral theory predicts that the rate of evolution is inversely related to the level of functional constraint. Therefore, selection against the use of non-preferred codons among those coding for the same amino acid should result in lower rates of synonymous substitution as compared with sites not subject to such selection pressures. However, reliably measuring the extent of such a relationship is problematic, as estimates of synonymous rates are sensitive to our assumptions about the process of molecular evolution. Previous studies showed the importance of accounting for unequal codon frequencies, in particular when synonymous codon usage is highly biased. Yet, unequal codon frequencies can be modeled in different ways, making different assumptions about the mutation process. Here we conduct a simulation study to evaluate two different ways of modeling uneven codon frequencies and show that both model parameterizations can have a dramatic impact on rate estimates and affect biological conclusions about genome evolution. We reanalyze three large data sets to demonstrate the relevance of our results to empirical data analysis.

Canine parvovirus type 2 (CPV-2) and Feline panleukopenia virus (FPV) codon bias analysis reveals a progressive adaptation to the new niche after the host jump.

PubMed

Franzo, Giovanni; Tucciarone, Claudia Maria; Cecchinato, Mattia; Drigo, Michele

2017-09-01

Based on virus dependence from host cell machinery, their codon usage is expected to show a strong relation with the host one. Even if this association has been stated, especially for bacteria viruses, the linkage is considered to be less consistent for more complex organisms and a codon bias adaptation after host jump has never been proven. Canine parvovirus type 2 (CPV-2) was selected as a model because it represents a well characterized case of host jump, originating from Feline panleukopenia virus (FPV). The current study demonstrates that the adaptation to specific tissue and host codon bias affected CPV-2 evolution. Remarkably, FPV and CPV-2 showed a higher closeness toward the codon bias of the tissues they display the higher tropism for. Moreover, after the host jump, a clear and significant trend was evidenced toward a reduction in the distance between CPV-2 and the dog codon bias over time. This evidence was not confirmed for FPV, suggesting that an equilibrium has been reached during the prolonged virus-host co-evolution. Additionally, the presence of an intermediate pattern displayed by some strains infecting wild species suggests that these could have facilitated the host switch also by acting on codon bias. Copyright © 2017 Elsevier Inc. All rights reserved.

A mutated hygromycin resistance gene is functional in the n-alkane-assimilating yeast Candida tropicalis.

PubMed

Hara, A; Ueda, M; Misawa, S; Matsui, T; Furuhashi, K; Tanaka, A

2000-03-01

Development of a transformation system in the n-alkane-assimilating diploid yeast Candida tropicalis requires an antibiotic resistance gene in order to establish a selectable marker. The resistance gene for hygromycin B has often been used as a selectable marker in yeast transformation. However, C. tropicalis harboring the hygromycin resistance gene (HYG) was as sensitive to hygromycin B as the wild-type strain. Nine CTG codons were found in the ORF of the HYG gene. This codon has been reported to be translated as serine rather than leucine in Candida species. Analysis of the tRNA gene in C. tropicalis with the anticodon CAG [tRNA(CAG) gene], which is complementary to the codon CTG, showed that the sequence was highly similar to that of the C. maltosa tRNA(CAG) gene. In C. maltosa, the codon CTG is read as serine and not leucine. These results suggested that the HYG gene was not functional due to the nonuniversal usage of the CTG codon. Each of the nine CTG codons in the ORF of the HYG gene was changed to a CTC codon, which is read as leucine, by site-directed mutagenesis. When a plasmid containing the mutated HYG gene (HYG#) was constructed and introduced into C. tropicalis, hygromycin-resistant transformants were successfully obtained. This mutated hygromycin resistance gene may be useful for direct selection of C. tropicalis transformants.

Properties and determinants of codon decoding time distributions

PubMed Central

2014-01-01

Background Codon decoding time is a fundamental property of mRNA translation believed to affect the abundance, function, and properties of proteins. Recently, a novel experimental technology--ribosome profiling--was developed to measure the density, and thus the speed, of ribosomes at codon resolution. Specifically, this method is based on next-generation sequencing, which theoretically can provide footprint counts that correspond to the probability of observing a ribosome in this position for each nucleotide in each transcript. Results In this study, we report for the first time various novel properties of the distribution of codon footprint counts in five organisms, based on large-scale analysis of ribosomal profiling data. We show that codons have distinctive footprint count distributions. These tend to be preserved along the inner part of the ORF, but differ at the 5' and 3' ends of the ORF, suggesting that the translation-elongation stage actually includes three biophysical sub-steps. In addition, we study various basic properties of the codon footprint count distributions and show that some of them correlate with the abundance of the tRNA molecule types recognizing them. Conclusions Our approach emphasizes the advantages of analyzing ribosome profiling and similar types of data via a comparative genomic codon-distribution-centric view. Thus, our methods can be used in future studies related to translation and even transcription elongation. PMID:25572668

Analysis of base and codon usage by rubella virus.

PubMed

Zhou, Yumei; Chen, Xianfeng; Ushijima, Hiroshi; Frey, Teryl K

2012-05-01

Rubella virus (RUBV), a small, plus-strand RNA virus that is an important human pathogen, has the unique feature that the GC content of its genome (70%) is the highest (by 20%) among RNA viruses. To determine the effect of this GC content on genomic evolution, base and codon usage were analyzed across viruses from eight diverse genotypes of RUBV. Despite differences in frequency of codon use, the favored codons in the RUBV genome matched those in the human genome for 18 of the 20 amino acids, indicating adaptation to the host. Although usage patterns were conserved in corresponding genes in the diverse genotypes, within-genome comparison revealed that both base and codon usages varied regionally, particularly in the hypervariable region (HVR) of the P150 replicase gene. While directional mutation pressure was predominant in determining base and codon usage within most of the genome (with the strongest tendency being towards C's at third codon positions), natural selection was predominant in the HVR region. The GC content of this region was the highest in the genome (>80%), and it was not clear if selection at the nucleotide level accompanied selection at the amino acid level. Dinucleotide frequency analysis of the RUBV genome revealed that TpA usage was lower than expected, similar to mammalian genes; however, CpG usage was not suppressed, and TpG usage was not enhanced, as is the case in mammalian genes.

Global analysis of translation termination in E. coli.

PubMed

Baggett, Natalie E; Zhang, Yan; Gross, Carol A

2017-03-01

Terminating protein translation accurately and efficiently is critical for both protein fidelity and ribosome recycling for continued translation. The three bacterial release factors (RFs) play key roles: RF1 and 2 recognize stop codons and terminate translation; and RF3 promotes disassociation of bound release factors. Probing release factors mutations with reporter constructs containing programmed frameshifting sequences or premature stop codons had revealed a propensity for readthrough or frameshifting at these specific sites, but their effects on translation genome-wide have not been examined. We performed ribosome profiling on a set of isogenic strains with well-characterized release factor mutations to determine how they alter translation globally. Consistent with their known defects, strains with increasingly severe release factor defects exhibit increasingly severe accumulation of ribosomes over stop codons, indicative of an increased duration of the termination/release phase of translation. Release factor mutant strains also exhibit increased occupancy in the region following the stop codon at a significant number of genes. Our global analysis revealed that, as expected, translation termination is generally efficient and accurate, but that at a significant number of genes (≥ 50) the ribosome signature after the stop codon is suggestive of translation past the stop codon. Even native E. coli K-12 exhibits the ribosome signature suggestive of protein extension, especially at UGA codons, which rely exclusively on the reduced function RF2 variant of the K-12 strain for termination. Deletion of RF3 increases the severity of the defect. We unambiguously demonstrate readthrough and frameshifting protein extensions and their further accumulation in mutant strains for a few select cases. In addition to enhancing recoding, ribosome accumulation over stop codons disrupts attenuation control of biosynthetic operons, and may alter expression of some overlapping genes. Together, these functional alterations may either augment the protein repertoire or produce deleterious proteins.

Somatic mutations in cancer: Stochastic versus predictable.

PubMed

Gold, Barry

2017-02-01

The origins of human cancers remain unclear except for a limited number of potent environmental mutagens, such as tobacco and UV light, and in rare cases, familial germ line mutations that affect tumor suppressor genes or oncogenes. A significant component of cancer etiology has been deemed stochastic and correlated with the number of stem cells in a tissue, the number of times the stem cells divide and a low incidence of random DNA polymerase errors that occur during each cell division. While somatic mutations occur during each round of DNA replication, mutations in cancer driver genes are not stochastic. Out of a total of 2843 codons, 1031 can be changed to stop codons by a single base substitution in the tumor suppressor APC gene, which is mutated in 76% of colorectal cancers (CRC). However, the nonsense mutations, which comprise 65% of all the APC driver mutations in CRC, are not random: 43% occur at Arg CGA codons, although they represent <3% of the codons. In TP53, CGA codons comprise <3% of the total 393 codons but they account for 72% and 39% of the mutations in CRC and ovarian cancer OVC, respectively. This mutation pattern is consistent with the kinetically slow, but not stochastic, hydrolytic deamination of 5-methylcytosine residues at specific methylated CpG sites to afford T·G mismatches that lead to C→T transitions and stop codons at CGA. Analysis of nonsense mutations in CRC, OVC and a number of other cancers indicates the need to expand the predictable risk factors for cancer to include, in addition to random polymerase errors, the methylation status of gene body CGA codons in tumor suppressor genes. Copyright © 2017. Published by Elsevier B.V.

The Acheta domesticus Densovirus, Isolated from the European House Cricket, Has Evolved an Expression Strategy Unique among Parvoviruses▿†

PubMed Central

Liu, Kaiyu; Li, Yi; Jousset, Françoise-Xavière; Zadori, Zoltan; Szelei, Jozsef; Yu, Qian; Pham, Hanh Thi; Lépine, François; Bergoin, Max; Tijssen, Peter

2011-01-01

The Acheta domesticus densovirus (AdDNV), isolated from crickets, has been endemic in Europe for at least 35 years. Severe epizootics have also been observed in American commercial rearings since 2009 and 2010. The AdDNV genome was cloned and sequenced for this study. The transcription map showed that splicing occurred in both the nonstructural (NS) and capsid protein (VP) multicistronic RNAs. The splicing pattern of NS mRNA predicted 3 nonstructural proteins (NS1 [576 codons], NS2 [286 codons], and NS3 [213 codons]). The VP gene cassette contained two VP open reading frames (ORFs), of 597 (ORF-A) and 268 (ORF-B) codons. The VP2 sequence was shown by N-terminal Edman degradation and mass spectrometry to correspond with ORF-A. Mass spectrometry, sequencing, and Western blotting of baculovirus-expressed VPs versus native structural proteins demonstrated that the VP1 structural protein was generated by joining ORF-A and -B via splicing (splice II), eliminating the N terminus of VP2. This splice resulted in a nested set of VP1 (816 codons), VP3 (467 codons), and VP4 (429 codons) structural proteins. In contrast, the two splices within ORF-B (Ia and Ib) removed the donor site of intron II and resulted in VP2, VP3, and VP4 expression. ORF-B may also code for several nonstructural proteins, of 268, 233, and 158 codons. The small ORF-B contains the coding sequence for a phospholipase A2 motif found in VP1, which was shown previously to be critical for cellular uptake of the virus. These splicing features are unique among parvoviruses and define a new genus of ambisense densoviruses. PMID:21775445

Overcoming codon-usage bias in heterologous protein expression in Streptococcus gordonii.

PubMed

Lee, Song F; Li, Yi-Jing; Halperin, Scott A

2009-11-01

One of the limitations facing the development of Streptococcus gordonii into a successful vaccine vector is the inability of this bacterium to express high levels of heterologous proteins. In the present study, we have identified 12 codons deemed as rare codons in S. gordonii and seven other streptococcal species. tRNA genes encoding 10 of the 12 rare codons were cloned into a plasmid. The plasmid was transformed into strains of S. gordonii expressing the fusion protein SpaP/S1, the anti-complement receptor 1 (CR1) single-chain variable fragment (scFv) antibody, or the Toxoplasma gondii cyclophilin C18 protein. These three heterologous proteins contained high percentages of amino acids encoded by rare codons. The results showed that the production of SpaP/S1, anti-CR1 scFv and C18 increased by 2.7-, 120- and 10-fold, respectively, over the control strains. In contrast, the production of the streptococcal SpaP protein without the pertussis toxin S1 fragment was not affected by tRNA gene supplementation, indicating that the increased production of SpaP/S1 protein was due to the ability to overcome the limitation caused by rare codons required for the S1 fragment. The increase in anti-CR1 scFv production was also observed in Streptococcus mutans following tRNA gene supplementation. Collectively, the findings in the present study demonstrate for the first time, to the best of our knowledge, that codon-usage bias exists in Streptococcus spp. and the limitation of heterologous protein expression caused by codon-usage bias can be overcome by tRNA supplementation.

tRNAomics: tRNA gene copy number variation and codon use provide bioinformatic evidence of a new anticodon:codon wobble pair in a eukaryote

PubMed Central

Iben, James R.; Maraia, Richard J.

2012-01-01

tRNA genes are interspersed throughout eukaryotic DNA, contributing to genome architecture and evolution in addition to translation of the transcriptome. Codon use correlates with tRNA gene copy number in noncomplex organisms including yeasts. Synonymous codons impact translation with various outcomes, dependent on relative tRNA abundances. Availability of whole-genome sequences allowed us to examine tRNA gene copy number variation (tgCNV) and codon use in four Schizosaccharomyces species and Saccharomyces cerevisiae. tRNA gene numbers vary from 171 to 322 in the four Schizosaccharomyces despite very high similarity in other features of their genomes. In addition, we performed whole-genome sequencing of several related laboratory strains of Schizosaccharomyces pombe and found tgCNV at a cluster of tRNA genes. We examined for the first time effects of wobble rules on correlation of tRNA gene number and codon use and showed improvement for S. cerevisiae and three of the Schizosaccharomyces species. In contrast, correlation in Schizosaccharomyces japonicus is poor due to markedly divergent tRNA gene content, and much worsened by the wobble rules. In japonicus, some tRNA iso-acceptor genes are absent and others are greatly reduced relative to the other yeasts, while genes for synonymous wobble iso-acceptors are amplified, indicating wobble use not apparent in any other eukaryote. We identified a subset of japonicus-specific wobbles that improves correlation of codon use and tRNA gene content in japonicus. We conclude that tgCNV is high among Schizo species and occurs in related laboratory strains of S. pombe (and expectedly other species), and tRNAome-codon analyses can provide insight into species-specific wobble decoding. PMID:22586155

Efficient Coproduction of Mannanase and Cellulase by the Transformation of a Codon-Optimized Endomannanase Gene from Aspergillus niger into Trichoderma reesei.

PubMed

Sun, Xianhua; Xue, Xianli; Li, Mengzhu; Gao, Fei; Hao, Zhenzhen; Huang, Huoqing; Luo, Huiying; Qin, Lina; Yao, Bin; Su, Xiaoyun

2017-12-20

Cellulase and mannanase are both important enzyme additives in animal feeds. Expressing the two enzymes simultaneously within one microbial host could potentially lead to cost reductions in the feeding of animals. For this purpose, we codon-optimized the Aspergillus niger Man5A gene to the codon-usage bias of Trichoderma reesei. By comparing the free energies and the local structures of the nucleotide sequences, one optimized sequence was finally selected and transformed into the T. reesei pyridine-auxotrophic strain TU-6. The codon-optimized gene was expressed to a higher level than the original one. Further expressing the codon-optimized gene in a mutated T. reesei strain through fed-batch cultivation resulted in coproduction of cellulase and mannanase up to 1376 U·mL -1 and 1204 U·mL -1 , respectively.

Physical Model for the Evolution of the Genetic Code

NASA Astrophysics Data System (ADS)

Yamashita, Tatsuro; Narikiyo, Osamu

2011-12-01

Using the shape space of codons and tRNAs we give a physical description of the genetic code evolution on the basis of the codon capture and ambiguous intermediate scenarios in a consistent manner. In the lowest dimensional version of our description, a physical quantity, codon level is introduced. In terms of the codon levels two scenarios are typically classified into two different routes of the evolutional process. In the case of the ambiguous intermediate scenario we perform an evolutional simulation implemented cost selection of amino acids and confirm a rapid transition of the code change. Such rapidness reduces uncomfortableness of the non-unique translation of the code at intermediate state that is the weakness of the scenario. In the case of the codon capture scenario the survival against mutations under the mutational pressure minimizing GC content in genomes is simulated and it is demonstrated that cells which experience only neutral mutations survive.

Reassigning stop codons via translation termination: How a few eukaryotes broke the dogma.

PubMed

Alkalaeva, Elena; Mikhailova, Tatiana

2017-03-01

The genetic code determines how amino acids are encoded within mRNA. It is universal among the vast majority of organisms, although several exceptions are known. Variant genetic codes are found in ciliates, mitochondria, and numerous other organisms. All revealed genetic codes (standard and variant) have at least one codon encoding a translation stop signal. However, recently two new genetic codes with a reassignment of all three stop codons were revealed in studies examining the protozoa transcriptomes. Here, we discuss this finding and the recent studies of variant genetic codes in eukaryotes. We consider the possible molecular mechanisms allowing the use of certain codons as sense and stop signals simultaneously. The results obtained by studying these amazing organisms represent a new and exciting insight into the mechanism of stop codon decoding in eukaryotes. Also see the video abstract here. © 2017 WILEY Periodicals, Inc.

BATTLE: Biomarker-Based Approaches of Targeted Therapy for Lung Cancer Elimination

DTIC Science & Technology

2008-04-01

although a grade 3 neutropenia was dose-limiting in one importance. Th th ubstrate of the CYP3A4 isoenzyme and P-gp. Its metabolism is sensitive to...tratification in clinis Molecular Pathway Biomarkers Type of Analysis EGFR EGFR Mutation ( exons 18 to 21) DNA sequencing EGFR Increased Copy Number...polysomy/am 1plification) DNA FISH K-Ras/B-Raf K-RAS Mutation (codons 12,13, 61) DNA sequencing B-RAF Mutations ( exons 11 and 15) DNA sequencing

PCR-RFLP to Detect Codon 248 Mutation in Exon 7 of "p53" Tumor Suppressor Gene

ERIC Educational Resources Information Center

Ouyang, Liming; Ge, Chongtao; Wu, Haizhen; Li, Suxia; Zhang, Huizhan

2009-01-01

Individual genome DNA was extracted fast from oral swab and followed up with PCR specific for codon 248 of "p53" tumor suppressor gene. "Msp"I restriction mapping showed the G-C mutation in codon 248, which closely relates to cancer susceptibility. Students learn the concepts, detection techniques, and research significance of point mutations or…

Codon influence on protein expression in E. coli correlates with mRNA levels

PubMed Central

Boël, Grégory; Wong, Kam-Ho; Su, Min; Luff, Jon; Valecha, Mayank; Everett, John K.; Acton, Thomas B.; Xiao, Rong; Montelione, Gaetano T.; Aalberts, Daniel P.; Hunt, John F.

2016-01-01

Degeneracy in the genetic code, which enables a single protein to be encoded by a multitude of synonymous gene sequences, has an important role in regulating protein expression, but substantial uncertainty exists concerning the details of this phenomenon. Here we analyze the sequence features influencing protein expression levels in 6,348 experiments using bacteriophage T7 polymerase to synthesize messenger RNA in Escherichia coli. Logistic regression yields a new codon-influence metric that correlates only weakly with genomic codon-usage frequency, but strongly with global physiological protein concentrations and also mRNA concentrations and lifetimes in vivo. Overall, the codon content influences protein expression more strongly than mRNA-folding parameters, although the latter dominate in the initial ~16 codons. Genes redesigned based on our analyses are transcribed with unaltered efficiency but translated with higher efficiency in vitro. The less efficiently translated native sequences show greatly reduced mRNA levels in vivo. Our results suggest that codon content modulates a kinetic competition between protein elongation and mRNA degradation that is a central feature of the physiology and also possibly the regulation of translation in E. coli. PMID:26760206

On the possible origin and evolution of the genetic code

NASA Technical Reports Server (NTRS)

Jukes, T. H.

1974-01-01

The genetic code is examined for indications of possible preceding codes that existed during early evolution. Eight of the 20 amino acids are coded by 'quartets' of codons with fourfold degeneracy, and 16 such quartets can exist, so that an earlier code could have provided for 15 or 16 amino acids, rather than 20. If twofold degeneracy is postulated for the first position of the codon, there could have been ten amino acids in the code. It is speculated that these may have been phenylalanine, valine, proline, alanine, histidine, glutamine, glutanic acid, aspartic acid, cysteine and glycine. There is a notable deficiency of arginine in proteins, despite the fact that it has six codons. Simultaneously, there is more lysine in proteins than would be expected from its two codons, if the four bases in mRNA are equiprobable and are arranged randomly. It is speculated that arginine is an 'intruder' into the genetic code, and that it may have displayed another amino acid such as ornithine, or may even have displayed lysine from some of its previous codon assignments. As a result, natural selection has favored lysine against the fact that it has only two codons.

Demonstration of GTG as an alternative initiation codon for the serpin endopin 2B-2.

PubMed

Hwang, Shin-Rong; Garza, Christina Z; Wegrzyn, Jill L; Hook, Vivian Y H

2005-02-18

This study demonstrates GTG as a novel, alternative initiation codon for translation of bovine endopin 2B-2, a serpin protease inhibitor. Molecular cDNA cloning revealed the endopin 2B-1 and endopin 2B-2 isoforms that are predicted to inhibit papain and elastase. Notably, GTG was demonstrated as the initiation codon for endopin 2B-2, whereas endopin 2B-1 possesses ATG as its initiation codon. GTG mediated in vitro translation of 46kDa endopin 2B-2. GTG also mediated translation of EGFP by in vitro translation and by expression in mammalian cells. Notably, mutagenesis of GTG to GTC resulted in the absence of EGFP expression in cells. GTG produced a lower level of protein expression compared to ATG. The use of GTG as an initiation codon to direct translation of endopin 2B, as well as the heterologous protein EGFP, demonstrates the role of GTG in the regulation of mRNA translation in mammalian cells. Significantly, further analyses of mammalian genomes based on GTG as an alternative initiation codon may predict new candidate gene products expressed by mammalian and human genomes.

Nonneutral GC3 and retroelement codon mimicry in Phytophthora.

PubMed

Jiang, Rays H Y; Govers, Francine

2006-10-01

Phytophthora is a genus entirely comprised of destructive plant pathogens. It belongs to the Stramenopila, a unique branch of eukaryotes, phylogenetically distinct from plants, animals, or fungi. Phytophthora genes show a strong preference for usage of codons ending with G or C (high GC3). The presence of high GC3 in genes can be utilized to differentiate coding regions from noncoding regions in the genome. We found that both selective pressure and mutation bias drive codon bias in Phytophthora. Indicative for selection pressure is the higher GC3 value of highly expressed genes in different Phytophthora species. Lineage specific GC increase of noncoding regions is reminiscent of whole-genome mutation bias, whereas the elevated Phytophthora GC3 is primarily a result of translation efficiency-driven selection. Heterogeneous retrotransposons exist in Phytophthora genomes and many of them vary in their GC content. Interestingly, the most widespread groups of retroelements in Phytophthora show high GC3 and a codon bias that is similar to host genes. Apparently, selection pressure has been exerted on the retroelement's codon usage, and such mimicry of host codon bias might be beneficial for the propagation of retrotransposons.

[Identifying and sequence analysis of HLA-B*2736].

PubMed

Li, Zhen; Zou, Hong-Yan; Shao, Chao-Peng; Tang, Si; Wang, Da-Ming; Cheng, Liang-Hong

2007-11-01

An unknown HLA-B allele which was similar to HLA-B*270401 was detected by FLOW-SSOPCR-SSP and heterozygous sequence-based typing (SBT) in Chinese Han individual. Its anomalous patterns suggested the possible presence of new allele. Amplifying exon 2-5(include intron 2-4) of the HLA-B*27 allele separately by using allele-specific primers and sequencing in both directions. Identifying the difference between the novel B*27 allele and B*270401. The sequence of novel B*27 from exon 2 to partial exon 5 is 1 815 bp. There are 10 nt changes from B*270401 in exon 3-4, at nt634where A-->C(codon130 AGC-->CGC, 130 S-->R); nt670 where A-->T (codon142 ACC-->TCC, 142 T-->S); nt683 where G-->T (codon146 TGG-->TTG, 146 W-->L); nt698 where A-->T (codon151 GAG-->GTG, 151 E-->V); nt774 where G-->C (codon176 GAG-->GAC, 176 E-->D); nt776 where C-->A (codon177 ACG-->AAG, 177 T-->K); nt781 where C-->G (codon179 CAG-->GAG, 179Q-->E); nt789 where G-->T (codon181 GCG-->GCT) resulting no coding change; nt1438 where C-->T (codon206 GGC-->GGT) resulting no coding change; nt1449 where G-->C (codon210 GGG-->GCG, 210G-->A). In IMGT/HLA database, only three alleles (B*270502/2706/2732) have sequences of introns. The same sequence in intron 2 showed homology between the novel HLA-B*27 allele and B*2706, but their homology could not be supported in intron 3-4. Comparing the sequence of the novel B*27 allele in intron 3 and 4 with B*27 group, it showed there are three mutations at nt106 C-->G, nt179 G-->A, nt536 G-->A and one deletion at nt168 in intron 3 and one mutations at nt82 T-->C in intron 4, but the sequence of the novel B*27 allele in intron 3 and 4 was all the same to B*070201. The sequence was submitted to Gen-Bank and the accession number was DQ915176. The allele has been confirmed as an extension of B*2736 by the WHO Nomenclature committee in November 2006.

Zika Virus Attenuation by Codon Pair Deoptimization Induces Sterilizing Immunity in Mouse Models.

PubMed

Li, Penghui; Ke, Xianliang; Wang, Ting; Tan, Zhongyuan; Luo, Dan; Miao, Yuanjiu; Sun, Jianhong; Zhang, Yuan; Liu, Yan; Hu, Qinxue; Xu, Fuqiang; Wang, Hanzhong; Zheng, Zhenhua

2018-06-20

Zika virus (ZIKV) infection during the large epidemics in the Americas is related to congenital abnormities or fetal demise. To date, there is no vaccine, antiviral drug, or other modality available to prevent or treat Zika virus infection. Here we designed novel live attenuated ZIKV vaccine candidates using a codon pair deoptimization strategy. Three codon pair-deoptimized ZIKVs (Min E, Min NS1, and Min E+NS1) were de novo synthesized, and recovered by reverse genetics, containing large amounts of underrepresented codon pairs in E gene and/or NS1 gene. Amino acid sequence was 100% unchanged. The codon pair-deoptimized variants had decreased replication fitness in Vero cells (Min NS1 ≫ Min E > Min E+NS1), replicated more efficiently in insect cells than in mammalian cells, and demonstrated diminished virulence in a mouse model. In particular, Min E+NS1, the most restrictive variant, induced sterilizing immunity with a robust neutralizing antibody titer, and a single immunization achieved complete protection against lethal challenge and vertical ZIKV transmission during pregnancy. More importantly, due to the numerous synonymous substitutions in the codon pair-deoptimized strains, reversion to wild-type virulence through gradual nucleotide sequence mutations is unlikely. Our results collectively demonstrate that ZIKV can be effectively attenuated by codon pair deoptimization, highlighting the potential of Min E+NS1 as a safe vaccine candidate to prevent ZIKV infections. IMPORTANCE Due to unprecedented epidemics of Zika virus (ZIKV) across the Americas and the unexpected clinical symptoms including Guillain-Barré syndrome, microcephaly and other birth defects in human, there is an urgent need for ZIKV vaccine development. Here, we provided the first attenuated versions of ZIKV with two important genes (E and/or NS1) that were subjected to codon pair deoptimization. Compared to parental ZIKV, the codon pair-deoptimized ZIKVs were mammalian-attenuated, and preferred insect to mammalian Cells. Min E+NS1, the most restrictive variant, induced sterilizing immunity with a robust neutralizing antibody titer, and achieved complete protection against lethal challenge and vertical virus transmission during pregnancy. More importantly, the massive synonymous mutational approach made it impossible to revert to wild-type virulence. Our results have proven the feasibility of codon pair deoptimization as a strategy to develop live-attenuated vaccine candidates against flavivirues like ZIKV, Japanese encephalitis virus and West Nile virus. Copyright © 2018 American Society for Microbiology.

Introduction of a point mutation into the mouse genome by homologous recombination in embryonic stem cells using a replacement type vector with a selectable marker.

PubMed

Rubinstein, M; Japón, M A; Low, M J

1993-06-11

The introduction of small mutations instead of null alleles into the mouse genome has broad applications to the study of protein structure-function relationships and the creation of animal models of human genetic diseases. To test a simple mutational strategy we designed a targeting vector for the mouse proopiomelanocortin (POMC) gene containing a single nucleotide insertion that converts the initial tyrosine codon of beta-endorphin 1-31 to a premature translational termination codon and introduces a unique Hpal endonuclease restriction site. The targeting vector also contains a neo cassette immediately 3' to the last POMC exon and a herpes simplex virus thymidine kinase cassette to allow positive and negative selection. Homologous recombination occurred at a frequency of 1/30 clones of electroporated embryonic stem cells selected in G418 and gancyclovir. 10/11 clones identified initially by a polymerase chain reaction (PCR) strategy had the predicted structure without evidence of concatemer formation by Southern blot analysis. We used a combination of Hpa I digestion of PCR amplified fragments and direct nucleotide sequencing to further confirm that the point mutation was retained in 9/10 clones. The POMC gene was transcriptionally silent in embryonic stem cells and the targeted allele was not activated by the downstream phosphoglycerate kinase-1 promoter that transcribed the neo gene. Under the electroporation conditions used, we have demonstrated that a point mutation can be introduced with high efficiency and precision into the POMC gene using a replacement type vector containing a retained selectable marker without affecting expression of the allele in the embryonic stem cells. A similar strategy may be useful for a wide range of genes.

Introduction of a point mutation into the mouse genome by homologous recombination in embryonic stem cells using a replacement type vector with a selectable marker.

PubMed Central

Rubinstein, M; Japón, M A; Low, M J

1993-01-01

The introduction of small mutations instead of null alleles into the mouse genome has broad applications to the study of protein structure-function relationships and the creation of animal models of human genetic diseases. To test a simple mutational strategy we designed a targeting vector for the mouse proopiomelanocortin (POMC) gene containing a single nucleotide insertion that converts the initial tyrosine codon of beta-endorphin 1-31 to a premature translational termination codon and introduces a unique Hpal endonuclease restriction site. The targeting vector also contains a neo cassette immediately 3' to the last POMC exon and a herpes simplex virus thymidine kinase cassette to allow positive and negative selection. Homologous recombination occurred at a frequency of 1/30 clones of electroporated embryonic stem cells selected in G418 and gancyclovir. 10/11 clones identified initially by a polymerase chain reaction (PCR) strategy had the predicted structure without evidence of concatemer formation by Southern blot analysis. We used a combination of Hpa I digestion of PCR amplified fragments and direct nucleotide sequencing to further confirm that the point mutation was retained in 9/10 clones. The POMC gene was transcriptionally silent in embryonic stem cells and the targeted allele was not activated by the downstream phosphoglycerate kinase-1 promoter that transcribed the neo gene. Under the electroporation conditions used, we have demonstrated that a point mutation can be introduced with high efficiency and precision into the POMC gene using a replacement type vector containing a retained selectable marker without affecting expression of the allele in the embryonic stem cells. A similar strategy may be useful for a wide range of genes. Images PMID:8392702

Genome-wide A-to-I RNA editing in fungi independent of ADAR enzymes

PubMed Central

Liu, Huiquan; Wang, Qinhu; He, Yi; Chen, Lingfeng; Hao, Chaofeng; Jiang, Cong; Li, Yang; Dai, Yafeng; Kang, Zhensheng; Xu, Jin-Rong

2016-01-01

Yeasts and filamentous fungi do not have adenosine deaminase acting on RNA (ADAR) orthologs and are believed to lack A-to-I RNA editing, which is the most prevalent editing of mRNA in animals. However, during this study with the PUK1 (FGRRES_01058) pseudokinase gene important for sexual reproduction in Fusarium graminearum, we found that two tandem stop codons, UA1831GUA1834G, in its kinase domain were changed to UG1831GUG1834G by RNA editing in perithecia. To confirm A-to-I editing of PUK1 transcripts, strand-specific RNA-seq data were generated with RNA isolated from conidia, hyphae, and perithecia. PUK1 was almost specifically expressed in perithecia, and 90% of transcripts were edited to UG1831GUG1834G. Genome-wide analysis identified 26,056 perithecium-specific A-to-I editing sites. Unlike those in animals, 70.5% of A-to-I editing sites in F. graminearum occur in coding regions, and more than two-thirds of them result in amino acid changes, including editing of 69 PUK1-like pseudogenes with stop codons in ORFs. PUK1 orthologs and other pseudogenes also displayed stage-specific expression and editing in Neurospora crassa and F. verticillioides. Furthermore, F. graminearum differs from animals in the sequence preference and structure selectivity of A-to-I editing sites. Whereas A's embedded in RNA stems are targeted by ADARs, RNA editing in F. graminearum preferentially targets A's in hairpin loops, which is similar to the anticodon loop of tRNA targeted by adenosine deaminases acting on tRNA (ADATs). Overall, our results showed that A-to-I RNA editing occurs specifically during sexual reproduction and mainly in the coding regions in filamentous ascomycetes, involving adenosine deamination mechanisms distinct from metazoan ADARs. PMID:26934920

The high-level expression of human tissue plasminogen activator in the milk of transgenic mice with hybrid gene locus strategy.

PubMed

Zhou, Yanrong; Lin, Yanli; Wu, Xiaojie; Xiong, Fuyin; Lv, Yuemeng; Zheng, Tao; Huang, Peitang; Chen, Hongxing

2012-02-01

Transgene expression for the mammary gland bioreactor aimed at producing recombinant proteins requires optimized expression vector construction. Previously we presented a hybrid gene locus strategy, which was originally tested with human lactoferrin (hLF) as target transgene, and an extremely high-level expression of rhLF ever been achieved as to 29.8 g/l in mice milk. Here to demonstrate the broad application of this strategy, another 38.4 kb mWAP-htPA hybrid gene locus was constructed, in which the 3-kb genomic coding sequence in the 24-kb mouse whey acidic protein (mWAP) gene locus was substituted by the 17.4-kb genomic coding sequence of human tissue plasminogen activator (htPA), exactly from the start codon to the end codon. Corresponding five transgenic mice lines were generated and the highest expression level of rhtPA in the milk attained as to 3.3 g/l. Our strategy will provide a universal way for the large-scale production of pharmaceutical proteins in the mammary gland of transgenic animals.

The Rift Valley fever accessory proteins NSm and P78/NSm-GN are distinct determinants of virus propagation in vertebrate and invertebrate hosts

PubMed Central

Kreher, Felix; Tamietti, Carole; Gommet, Céline; Guillemot, Laurent; Ermonval, Myriam; Failloux, Anna-Bella; Panthier, Jean-Jacques; Bouloy, Michèle; Flamand, Marie

2014-01-01

Rift Valley fever virus (RVFV) is an enzootic virus circulating in Africa that is transmitted to its vertebrate host by a mosquito vector and causes severe clinical manifestations in humans and ruminants. RVFV has a tripartite genome of negative or ambisense polarity. The M segment contains five in-frame AUG codons that are alternatively used for the synthesis of two major structural glycoproteins, GN and GC, and at least two accessory proteins, NSm, a 14-kDa cytosolic protein, and P78/NSm-GN, a 78-kDa glycoprotein. To determine the relative contribution of P78 and NSm to RVFV infectivity, AUG codons were knocked out to generate mutant viruses expressing various sets of the M-encoded proteins. We found that, in the absence of the second AUG codon used to express NSm, a 13-kDa protein corresponding to an N-terminally truncated form of NSm, named NSm′, was synthesized from AUG 3. None of the individual accessory proteins had any significant impact on RVFV virulence in mice. However, a mutant virus lacking both NSm and NSm′ was strongly attenuated in mice and grew to reduced titers in murine macrophages, a major target cell type of RVFV. In contrast, P78 was not associated with reduced viral virulence in mice, yet it appeared as a major determinant of virus dissemination in mosquitoes. This study demonstrates how related accessory proteins differentially contribute to RVFV propagation in mammalian and arthropod hosts. PMID:26038497

A Novel Method to Predict Highly Expressed Genes Based on Radius Clustering and Relative Synonymous Codon Usage.

PubMed

Tran, Tuan-Anh; Vo, Nam Tri; Nguyen, Hoang Duc; Pham, Bao The

2015-12-01

Recombinant proteins play an important role in many aspects of life and have generated a huge income, notably in the industrial enzyme business. A gene is introduced into a vector and expressed in a host organism-for example, E. coli-to obtain a high productivity of target protein. However, transferred genes from particular organisms are not usually compatible with the host's expression system because of various reasons, for example, codon usage bias, GC content, repetitive sequences, and secondary structure. The solution is developing programs to optimize for designing a nucleotide sequence whose origin is from peptide sequences using properties of highly expressed genes (HEGs) of the host organism. Existing data of HEGs determined by practical and computer-based methods do not satisfy for qualifying and quantifying. Therefore, the demand for developing a new HEG prediction method is critical. We proposed a new method for predicting HEGs and criteria to evaluate gene optimization. Codon usage bias was weighted by amplifying the difference between HEGs and non-highly expressed genes (non-HEGs). The number of predicted HEGs is 5% of the genome. In comparison with Puigbò's method, the result is twice as good as Puigbò's one, in kernel ratio and kernel sensitivity. Concerning transcription/translation factor proteins (TF), the proposed method gives low TF sensitivity, while Puigbò's method gives moderate one. In summary, the results indicated that the proposed method can be a good optional applying method to predict optimized genes for particular organisms, and we generated an HEG database for further researches in gene design.

Decoding Mechanisms by which Silent Codon Changes Influence Protein Biogenesis and Function

PubMed Central

Bali, Vedrana; Bebok, Zsuzsanna

2015-01-01

Scope Synonymous codon usage has been a focus of investigation since the discovery of the genetic code and its redundancy. The occurrences of synonymous codons vary between species and within genes of the same genome, known as codon usage bias. Today, bioinformatics and experimental data allow us to compose a global view of the mechanisms by which the redundancy of the genetic code contributes to the complexity of biological systems from affecting survival in prokaryotes, to fine tuning the structure and function of proteins in higher eukaryotes. Studies analyzing the consequences of synonymous codon changes in different organisms have revealed that they impact nucleic acid stability, protein levels, structure and function without altering amino acid sequence. As such, synonymous mutations inevitably contribute to the pathogenesis of complex human diseases. Yet, fundamental questions remain unresolved regarding the impact of silent mutations in human disorders. In the present review we describe developments in this area concentrating on mechanisms by which synonymous mutations may affect protein function and human health. Purpose This synopsis illustrates the significance of synonymous mutations in disease pathogenesis. We review the different steps of gene expression affected by silent mutations, and assess the benefits and possible harmful effects of codon optimization applied in the development of therapeutic biologics. Physiological and medical relevance Understanding mechanisms by which synonymous mutations contribute to complex diseases such as cancer, neurodegeneration and genetic disorders, including the limitations of codon-optimized biologics, provides insight concerning interpretation of silent variants and future molecular therapies. PMID:25817479

Codon Usage Patterns of Tyrosinase Genes in Clonorchis sinensis.

PubMed

Bae, Young-An

2017-04-01

Codon usage bias (CUB) is a unique property of genomes and has contributed to the better understanding of the molecular features and the evolution processes of particular gene. In this study, genetic indices associated with CUB, including relative synonymous codon usage and effective numbers of codons, as well as the nucleotide composition, were investigated in the Clonorchis sinensis tyrosinase genes and their platyhelminth orthologs, which play an important role in the eggshell formation. The relative synonymous codon usage patterns substantially differed among tyrosinase genes examined. In a neutrality analysis, the correlation between GC 12 and GC 3 was statistically significant, and the regression line had a relatively gradual slope (0.218). NC-plot, i.e., GC 3 vs effective number of codons (ENC), showed that most of the tyrosinase genes were below the expected curve. The codon adaptation index (CAI) values of the platyhelminth tyrosinases had a narrow distribution between 0.685/0.714 and 0.797/0.837, and were negatively correlated with their ENC. Taken together, these results suggested that CUB in the tyrosinase genes seemed to be basically governed by selection pressures rather than mutational bias, although the latter factor provided an additional force in shaping CUB of the C. sinensis and Opisthorchis viverrini genes. It was also apparent that the equilibrium point between selection pressure and mutational bias is much more inclined to selection pressure in highly expressed C. sinensis genes, than in poorly expressed genes.

Ribosome stalling and peptidyl-tRNA drop-off during translational delay at AGA codons

PubMed Central

Cruz-Vera, Luis Rogelio; Magos-Castro, Marco Antonio; Zamora-Romo, Efraín; Guarneros, Gabriel

2004-01-01

Minigenes encoding the peptide Met–Arg–Arg have been used to study the mechanism of toxicity of AGA codons proximal to the start codon or prior to the termination codon in bacteria. The codon sequences of the ‘mini-ORFs’ employed were initiator, combinations of AGA and CGA, and terminator. Both, AGA and CGA are low-usage Arg codons in ORFs of Escherichia coli but, whilst AGA is translated by the scarce tRNAArg4, CGA is recognized by the abundant tRNAArg2. Overexpression of minigenes harbouring AGA in the third position, next to a termination codon, was deleterious to the cell and led to the accumulation of peptidyl-tRNAArg4 and of the peptidyl-tRNA cognate to the preceding CGA or AGA Arg triplet. The minigenes carrying CGA in the third position were not toxic. Minigene-mediated toxicity and peptidyl-tRNA accumulation were suppressed by overproduction of tRNAArg4 but not by overproduction of peptidyl-tRNA hydrolase, an enzyme that is only active on substrates that have been released from the ribosome. Consistent with these findings, peptidyl-tRNAArg4 was identified to be mainly associated with ribosomes in a stand-by complex. These and previous results support the hypothesis that the primary mechanism of inhibition of protein synthesis by AGA triplets in pth+ cells involves sequestration of tRNAs as peptidyl-tRNA on the stalled ribosome. PMID:15317870

Codon optimisation to improve expression of a Mycobacterium avium ssp. paratuberculosis-specific membrane-associated antigen by Lactobacillus salivarius.

PubMed

Johnston, Christopher; Douarre, Pierre E; Soulimane, Tewfik; Pletzer, Daniel; Weingart, Helge; MacSharry, John; Coffey, Aidan; Sleator, Roy D; O'Mahony, Jim

2013-06-01

Subunit and DNA-based vaccines against Mycobacterium avium ssp. paratuberculosis (MAP) attempt to overcome inherent issues associated with whole-cell formulations. However, these vaccines can be hampered by poor expression of recombinant antigens from a number of disparate hosts. The high G+C content of MAP invariably leads to a codon bias throughout gene expression. To investigate if the codon bias affects recombinant MAP antigen expression, the open reading frame of a MAP-specific antigen MptD (MAP3733c) was codon optimised for expression against a Lactobacillus salivarius host. Of the total 209 codons which constitute MAP3733c, 172 were modified resulting in a reduced G+C content from 61% for the native gene to 32.7% for the modified form. Both genes were placed under the transcriptional control of the PnisA promoter; allowing controlled heterologous expression in L. salivarius. Expression was monitored using fluorescence microscopy and microplate fluorometry via GFP tags translationally fused to the C-termini of the two MptD genes. A > 37-fold increase in expression was observed for the codon-optimised MAP3733synth variant over the native gene. Due to the low cost and improved expression achieved, codon optimisation significantly improves the potential of L. salivarius as an oral vaccine stratagem against Johne's disease. © 2013 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved.

The PEPvIII-KLH (CDX-110) vaccine in glioblastoma multiforme patients.

PubMed

Heimberger, Amy B; Sampson, John H

2009-08-01

Conventional therapies for glioblastoma multiforme (GBM) fail to target tumor cells exclusively, resulting in non-specific toxicity. Immune targeting of tumor-specific mutations may allow for more precise eradication of neoplastic cells. EGFR variant III (EGFRvIII) is a tumor-specific mutation that is widely expressed in GBM and other neoplasms and its expression enhances tumorigenicity. This in-frame deletion mutation splits a codon, resulting in a novel glycine at the fusion junction producing a tumor-specific epitope target for cellular or humoral immunotherapy. We have previously shown that vaccination with a peptide that spans the EGFRvIII fusion junction (PEPvIII-KLH/CDX-110) is an efficacious immunotherapy in syngeneic murine models. In this review, we summarize our results in GBM patients targeting this mutation in multiple, multi-institutional Phase II immunotherapy trials. These trials demonstrated that a selected population of GBM patients who received vaccines targeting EGFRvIII had an unexpectedly long survival time. Further therapeutic strategies and potential pitfalls of using this approach are discussed.

Inspections of Interstate Commercial Vehicles 1994

DOT National Transportation Integrated Search

1974-01-01

The objective of this effort was to complete the development of the computer simulation model SCOT (Simulation of Corridor Traffic) designed to represent traffic flow on an urban grid-freeway integrated highway system by simulating an existing system...

Cambial history of Scots pine trees (Pinus Sylvestris) prior and after the Chernobyl accident as encoded in the xylem.

PubMed

Tulik, M

2001-08-01

Studies were carried out on wood samples collected in October 1997 from breast height of Scots pine trees (Pinus sylvestris) from site located 5 km south from the Chernobyl nuclear power plant. The radioactive contamination at the site was 3.7x10(5) kBq m(-2). These samples of secondary wood were used as an archive of information about the dynamics of a meristematic tissue cambium affected by ionising radiation from the Chernobyl reactor accident. The results show that frequency of the cambial cells events like anticlinal divisions, intrusive growth and cells elimination, was after the Chernobyl accident, about three times higher in comparison to preceding years. The most interesting finding was that after irradiation the length of tracheids increased. This increase is interpreted as an effect of intracambial competition among cells in the initial layer.

Wood anatomical parameters of lowland European oak and Scots pine as proxies for climate reconstructions

NASA Astrophysics Data System (ADS)

Balanzategui, Daniel; Heußner, Karl-Uwe; Wazny, Tomasz; Helle, Gerd; Heinrich, Ingo

2017-04-01

Tree-ring based temperature reconstructions from the temperate lowlands worldwide are largely missing due to diffuse climate signals so far found in tree-ring widths. This motivated us to concentrate our efforts on the wood anatomies of two common European tree species, the European oak (Quercus robur) and Scots pine (Pinus sylvestris). We combined core samples of living trees with archaeological wood from northern Germany and Poland. We measured approx. 46,000 earlywood oak vessels of 34 trees covering the period AD 1500 to 2016 and approx. 7.5 million pine tracheid cells of 41 trees covering the period AD 1300 to 2010. First climate growth analyses indicate that both oak earlywood vessel and pine tracheid parameters contain climate signals which are different and more significant than those found in tree-ring widths. Preliminary results will be presented and discussed at EGU for the first time.

Qualitative and quantitative determination of extractives in heartwood of Scots pine (Pinus sylvestris L.) by gas chromatography.

PubMed

Ekeberg, Dag; Flaete, Per-Otto; Eikenes, Morten; Fongen, Monica; Naess-Andresen, Carl Fredrik

2006-03-24

A method for quantitative determination of extractives from heartwood of Scots pine (Pinus sylvestris L.) using gas chromatography (GC) with flame ionization detection (FID) was developed. The limit of detection (LOD) was 0.03 mg/g wood and the linear range (r = 0.9994) was up to 10 mg/g with accuracy within +/- 10% and precision of 18% relative standard deviation. The identification of the extractives was performed using gas chromatography combined with mass spectrometry (GC-MS). The yields of extraction by Soxhlet were tested for solid wood, small particles and fine powder. Small particles were chosen for further analysis. This treatment gave good yields of the most important extractives: pinosylvin, pinosylvin monomethyl ether, resin acids and free fatty acids. The method is used to demonstrate the variation of these extractives across stems and differences in north-south direction.

Induction of discolored wood in Scots pine (Pinus sylvestris).

PubMed

Nilsson, Mikael; Wikman, Susanne; Eklund, Leif

2002-04-01

Induction of discolored wood in Scots pine (Pinus sylvestris L.) trees by treatment with ethylene, carbon dioxide, nitrogen (hypoxia) or wounding from early April to late September was investigated. All treatments induced formation of discolored wood upward and downward from the drill hole. The amount of discolored wood formed above the drill hole depended on the treatment in the following order: ethylene > carbon dioxide = nitrogen > wounding; and below the drill hole in the order: ethylene > carbon dioxide = nitrogen = wounding. Based on chemical analyses (HPLC/UV, GS/MS, LC/MS and 1H-NMR), discolored wood induced by wounding or treatment with ethylene or carbon dioxide showed compositional similarities to natural heartwood, whereas discolored wood induced by nitrogen treatment showed fewer similarities to natural heartwood. The results suggest that ethylene is an important factor controlling heartwood formation, although wounding and internal concentrations of carbon dioxide may also play a role.

Elevation of the Yields of Very Long Chain Polyunsaturated Fatty Acids via Minimal Codon Optimization of Two Key Biosynthetic Enzymes

PubMed Central

Zheng, Desong; Sun, Quanxi; Liu, Jiang; Li, Yaxiao; Hua, Jinping

2016-01-01

Eicosapentaenoic acid (EPA, 20:5Δ5,8,11,14,17) and Docosahexaenoic acid (DHA, 22:6Δ4,7,10,13,16,19) are nutritionally beneficial to human health. Transgenic production of EPA and DHA in oilseed crops by transferring genes originating from lower eukaryotes, such as microalgae and fungi, has been attempted in recent years. However, the low yield of EPA and DHA produced in these transgenic crops is a major hurdle for the commercialization of these transgenics. Many factors can negatively affect transgene expression, leading to a low level of converted fatty acid products. Among these the codon bias between the transgene donor and the host crop is one of the major contributing factors. Therefore, we carried out codon optimization of a fatty acid delta-6 desaturase gene PinD6 from the fungus Phytophthora infestans, and a delta-9 elongase gene, IgASE1 from the microalga Isochrysis galbana for expression in Saccharomyces cerevisiae and Arabidopsis respectively. These are the two key genes encoding enzymes for driving the first catalytic steps in the Δ6 desaturation/Δ6 elongation and the Δ9 elongation/Δ8 desaturation pathways for EPA/DHA biosynthesis. Hence expression levels of these two genes are important in determining the final yield of EPA/DHA. Via PCR-based mutagenesis we optimized the least preferred codons within the first 16 codons at their N-termini, as well as the most biased CGC codons (coding for arginine) within the entire sequences of both genes. An expression study showed that transgenic Arabidopsis plants harbouring the codon-optimized IgASE1 contained 64% more elongated fatty acid products than plants expressing the native IgASE1 sequence, whilst Saccharomyces cerevisiae expressing the codon optimized PinD6 yielded 20 times more desaturated products than yeast expressing wild-type (WT) PinD6. Thus the codon optimization strategy we developed here offers a simple, effective and low-cost alternative to whole gene synthesis for high expression of foreign genes in yeast and Arabidopsis. PMID:27433934

Analyses of frameshifting at UUU-pyrimidine sites.

PubMed

Schwartz, R; Curran, J F

1997-05-15

Others have recently shown that the UUU phenylalanine codon is highly frameshift-prone in the 3'(rightward) direction at pyrimidine 3'contexts. Here, several approaches are used to analyze frameshifting at such sites. The four permutations of the UUU/C (phenylalanine) and CGG/U (arginine) codon pairs were examined because they vary greatly in their expected frameshifting tendencies. Furthermore, these synonymous sites allow direct tests of the idea that codon usage can control frameshifting. Frameshifting was measured for these dicodons embedded within each of two broader contexts: the Escherichia coli prfB (RF2 gene) programmed frameshift site and a 'normal' message site. The principal difference between these contexts is that the programmed frameshift contains a purine-rich sequence upstream of the slippery site that can base pair with the 3'end of 16 S rRNA (the anti-Shine-Dalgarno) to enhance frameshifting. In both contexts frameshift frequencies are highest if the slippery tRNAPhe is capable of stable base pairing in the shifted reading frame. This requirement is less stringent in the RF2 context, as if the Shine-Dalgarno interaction can help stabilize a quasi-stable rephased tRNA:message complex. It was previously shown that frameshifting in RF2 occurs more frequently if the codon 3'to the slippery site is read by a rare tRNA. Consistent with that earlier work, in the RF2 context frameshifting occurs substantially more frequently if the arginine codon is CGG, which is read by a rare tRNA. In contrast, in the 'normal' context frameshifting is only slightly greater at CGG than at CGU. It is suggested that the Shine-Dalgarno-like interaction elevates frameshifting specifically during the pause prior to translation of the second codon, which makes frameshifting exquisitely sensitive to the rate of translation of that codon. In both contexts frameshifting increases in a mutant strain that fails to modify tRNA base A37, which is 3'of the anticodon. Thus, those base modifications may limit frameshifting at UUU codons. Finally, statistical analyses show that UUU Ynn dicodons are extremely rare in E.coli genes that have highly biased codon usage.

Analyses of frameshifting at UUU-pyrimidine sites.

PubMed Central

Schwartz, R; Curran, J F

1997-01-01

Others have recently shown that the UUU phenylalanine codon is highly frameshift-prone in the 3'(rightward) direction at pyrimidine 3'contexts. Here, several approaches are used to analyze frameshifting at such sites. The four permutations of the UUU/C (phenylalanine) and CGG/U (arginine) codon pairs were examined because they vary greatly in their expected frameshifting tendencies. Furthermore, these synonymous sites allow direct tests of the idea that codon usage can control frameshifting. Frameshifting was measured for these dicodons embedded within each of two broader contexts: the Escherichia coli prfB (RF2 gene) programmed frameshift site and a 'normal' message site. The principal difference between these contexts is that the programmed frameshift contains a purine-rich sequence upstream of the slippery site that can base pair with the 3'end of 16 S rRNA (the anti-Shine-Dalgarno) to enhance frameshifting. In both contexts frameshift frequencies are highest if the slippery tRNAPhe is capable of stable base pairing in the shifted reading frame. This requirement is less stringent in the RF2 context, as if the Shine-Dalgarno interaction can help stabilize a quasi-stable rephased tRNA:message complex. It was previously shown that frameshifting in RF2 occurs more frequently if the codon 3'to the slippery site is read by a rare tRNA. Consistent with that earlier work, in the RF2 context frameshifting occurs substantially more frequently if the arginine codon is CGG, which is read by a rare tRNA. In contrast, in the 'normal' context frameshifting is only slightly greater at CGG than at CGU. It is suggested that the Shine-Dalgarno-like interaction elevates frameshifting specifically during the pause prior to translation of the second codon, which makes frameshifting exquisitely sensitive to the rate of translation of that codon. In both contexts frameshifting increases in a mutant strain that fails to modify tRNA base A37, which is 3'of the anticodon. Thus, those base modifications may limit frameshifting at UUU codons. Finally, statistical analyses show that UUU Ynn dicodons are extremely rare in E.coli genes that have highly biased codon usage. PMID:9115369

Disruption of the Opal Stop Codon Attenuates Chikungunya Virus-Induced Arthritis and Pathology.

PubMed

Jones, Jennifer E; Long, Kristin M; Whitmore, Alan C; Sanders, Wes; Thurlow, Lance R; Brown, Julia A; Morrison, Clayton R; Vincent, Heather; Peck, Kayla M; Browning, Christian; Moorman, Nathaniel; Lim, Jean K; Heise, Mark T

2017-11-14

Chikungunya virus (CHIKV) is a mosquito-borne alphavirus responsible for several significant outbreaks of debilitating acute and chronic arthritis and arthralgia over the past decade. These include a recent outbreak in the Caribbean islands and the Americas that caused more than 1 million cases of viral arthralgia. Despite the major impact of CHIKV on global health, viral determinants that promote CHIKV-induced disease are incompletely understood. Most CHIKV strains contain a conserved opal stop codon at the end of the viral nsP3 gene. However, CHIKV strains that encode an arginine codon in place of the opal stop codon have been described, and deep-sequencing analysis of a CHIKV isolate from the Caribbean identified both arginine and opal variants within this strain. Therefore, we hypothesized that the introduction of the arginine mutation in place of the opal termination codon may influence CHIKV virulence. We tested this by introducing the arginine mutation into a well-characterized infectious clone of a CHIKV strain from Sri Lanka and designated this virus Opal524R. This mutation did not impair viral replication kinetics in vitro or in vivo Despite this, the Opal524R virus induced significantly less swelling, inflammation, and damage within the feet and ankles of infected mice. Further, we observed delayed induction of proinflammatory cytokines and chemokines, as well as reduced CD4 + T cell and NK cell recruitment compared to those in the parental strain. Therefore, the opal termination codon plays an important role in CHIKV pathogenesis, independently of effects on viral replication. IMPORTANCE Chikungunya virus (CHIKV) is a mosquito-borne alphavirus that causes significant outbreaks of viral arthralgia. Studies with CHIKV and other alphaviruses demonstrated that the opal termination codon within nsP3 is highly conserved. However, some strains of CHIKV and other alphaviruses contain mutations in the opal termination codon. These mutations alter the virulence of related alphaviruses in mammalian and mosquito hosts. Here, we report that a clinical isolate of a CHIKV strain from the recent outbreak in the Caribbean islands contains a mixture of viruses encoding either the opal termination codon or an arginine mutation. Mutating the opal stop codon to an arginine residue attenuates CHIKV-induced disease in a mouse model. Compared to infection with the opal-containing parental virus, infection with the arginine mutant causes limited swelling and inflammation, as well as dampened recruitment of immune mediators of pathology, including CD4 + T cells and NK cells. We propose that the opal termination codon plays an essential role in the induction of severe CHIKV disease. Copyright © 2017 Jones et al.

Selectivity of Pinus sylvestris extract and essential oil to estrogen-insensitive breast cancer cells Pinus sylvestris against cancer cells

PubMed Central

Hoai, Nguyen Thi; Duc, Ho Viet; Thao, Do Thi; Orav, Anne; Raal, Ain

2015-01-01

Background: So far, the anticancer action of pine tree extracts has mainly been shown for the species distributed widely around the Asian countries. Objective: Therefore, this study was performed to examine the potential cytotoxicity of Scots pine (Pinus sylvestris L.) native also to the European region and growing widely in Estonia. Materials and Methods: The cytotoxic activity of methanol extract and essential oil of Scots pine needles was determined by sulforhodamine B assay in different human cancer cell lines. Results: This needle extract was found to suppress the viability of several human cancer cell lines showing some selectivity to estrogen receptor negative breast cancer cells, MDA-MB-231(half maximal inhibitory concentration [IC50] 35 μg/ml) in comparison with estrogen receptor-positive breast cancer cells, MCF-7 (IC50 86 μg/ml). It is the strongest cytotoxic effect at all measured, thus far for the needles and leaves extracts derived from various pine species, and is also the first study comparing the anticancer effects of pine tree extracts on molecularly different human breast cancer cells. The essential oil showed the stronger cytotoxic effect to both negative and positive breast cancer cell lines (both IC50 29 μg/ml) than pine extract (IC50 42 and 80 μg/ml, respectively). Conclusion: The data from this report indicate that Scots pine needles extract and essential oil exhibits some potential as chemopreventive or chemotherapeutic agent for mammary tumors unresponsive to endocrine treatment. PMID:26664017

Fungal Infection Increases the Rate of Somatic Mutation in Scots Pine (Pinus sylvestris L.).

PubMed

Ranade, Sonali Sachin; Ganea, Laura-Stefana; Razzak, Abdur M; García Gil, M R

2015-01-01

Somatic mutations are transmitted during mitosis in developing somatic tissue. Somatic cells bearing the mutations can develop into reproductive (germ) cells and the somatic mutations are then passed on to the next generation of plants. Somatic mutations are a source of variation essential to evolve new defense strategies and adapt to the environment. Stem rust disease in Scots pine has a negative effect on wood quality, and thus adversely affects the economy. It is caused by the 2 most destructive fungal species in Scandinavia: Peridermium pini and Cronartium flaccidum. We studied nuclear genome stability in Scots pine under biotic stress (fungus-infected, 22 trees) compared to a control population (plantation, 20 trees). Stability was assessed as accumulation of new somatic mutations in 10 microsatellite loci selected for genotyping. Microsatellites are widely used as molecular markers in population genetics studies of plants, and are particularly used for detection of somatic mutations as their rate of mutation is of a much higher magnitude when compared with other DNA markers. We report double the rate of somatic mutation per locus in the fungus-infected trees (4.8×10(-3) mutations per locus), as compared to the controls (2.0×10(-3) mutations per locus) when individual samples were analyzed at 10 different microsatellite markers. Pearson's chi-squared test indicated a significant effect of the fungal infection which increased the number of mutations in the fungus-infected trees (χ(2) = 12.9883, df = 1, P = 0.0003134). © The American Genetic Association 2015. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Defoliating Insect Mass Outbreak Affects Soil N Fluxes and Tree N Nutrition in Scots Pine Forests.

PubMed

Grüning, Maren M; Simon, Judy; Rennenberg, Heinz; L-M-Arnold, Anne

2017-01-01

Biotic stress by mass outbreaks of defoliating pest insects does not only affect tree performance by reducing its photosynthetic capacity, but also changes N cycling in the soil of forest ecosystems. However, how insect induced defoliation affects soil N fluxes and, in turn, tree N nutrition is not well-studied. In the present study, we quantified N input and output fluxes via dry matter input, throughfall, and soil leachates. Furthermore, we investigated the effects of mass insect herbivory on tree N acquisition (i.e., organic and inorganic 15 N net uptake capacity of fine roots) as well as N pools in fine roots and needles in a Scots pine ( Pinus sylvestris L.) forest over an entire vegetation period. Plots were either infested by the nun moth ( Lymantria monacha L.) or served as controls. Our results show an increased N input by insect feces, litter, and throughfall at the infested plots compared to controls, as well as increased leaching of nitrate. However, the additional N input into the soil did not increase, but reduce inorganic and organic net N uptake capacity of Scots pine roots. N pools in the fine roots and needles of infested trees showed an accumulation of total N, amino acid-N, protein-N, and structural N in the roots and the remaining needles as a compensatory response triggered by defoliation. Thus, although soil N availability was increased via surplus N input, trees did not respond with an increased N acquisition, but rather invested resources into defense by accumulation of amino acid-N and protein-N as a survival strategy.

Seasonal and spatial variability of rainfall redistribution under Scots pine and Downy oak forests in Mediterranean conditions

NASA Astrophysics Data System (ADS)

Garcia-Estringana, Pablo; Latron, Jérôme; Molina, Antonio J.; Llorens, Pilar

2013-04-01

The large degree of temporal and spatial variability of throughfall input patterns may lead to significant changes in the volume of water that reach the soil in each location, and beyond in the hydrological response of forested hillslopes. To explore the role of vegetation in the temporal and spatial redistribution of rainfall in Mediterranean climatic conditions two contrasted stands were monitored. One is a Downy oak forest (Quercus pubescens) and the other is a Scots pine forest (Pinus sylvestris), both are located in the Vallcebre research catchments (NE Spain, 42° 12'N, 1° 49'E). These plots are representative of Mediterranean mountain areas with spontaneous afforestation by Scots pine as a consequence of the abandonment of agricultural terraces, formerly covered by Downy oaks. The monitoring design of each plot consists of a set of 20 automatic rain recorders and 40 automatic soil moisture probes located below the canopy. 100 hemispheric photographs of the canopy were used to place the instruments at representative locations (in terms of canopy cover) within the plot. Bulk rainfall, stemflow and meteorological conditions above the forest cover are also automatically recorded. Canopy cover as well as biometric characteristics of the plots are also regularly measured. This work presents the first results describing the variability of throughfall beneath each forest stand and compares the persistence of temporal patterns among stands, and for the oaks stand among the leafed and the leafless period. Furthermore, canopy structure, rainfall characteristics and meteorological conditions of rainfall events are evaluated as main drivers of throughfall redistribution.

Expression and purification of functional Clostridium perfringens alpha and epsilon toxins in Escherichia coli.

PubMed

Zhao, Yao; Kang, Lin; Gao, Shan; Zhou, Yang; Su, Libo; Xin, Wenwen; Su, Yuxin; Wang, Jinglin

2011-06-01

The alpha and epsilon toxins are 2 of the 4 major lethal toxins of the pathogen Clostridium perfringens. In this study, the expression of the epsilon toxin (etx) gene of C. perfringens was optimized by replacing rare codons with high-frequency codons, and the optimized gene was synthesized using overlapping PCR. Then, the etx gene or the alpha-toxin gene (cpa) was individually inserted into the pTIG-Trx expression vector with a hexahistidine tag and a thioredoxin (Trx) to facilitate their purification and induce the expression of soluble proteins. The recombinant alpha toxin (rCPA) and epsilon toxin (rETX) were highly expressed as soluble forms in the recipient Escherichia coli BL21 strain, respectively. The rCPA and rETX were purified using Ni(2+)-chelating chromatography and size-exclusion chromatography. And the entire purification process recovered about 40% of each target protein from the starting materials. The purified target toxins formed single band at about 42kDa (rCPA) or 31kDa (rETX) in sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and their functional activity was confirmed by bioactivity assays. We have shown that the production of large amounts of soluble and functional proteins by using the pTIG-Trx vector in E. coli is a good alternative for the production of native alpha and epsilon toxins and could also be useful for the production of other toxic proteins with soluble forms. Copyright © 2011 Elsevier Inc. All rights reserved.

Transformation of NIH3T3 Cells with Synthetic c‐Ha‐ras Genes

PubMed Central

Kamiya, Hiroyuki; Miura, Kazunobu; Ohtomo, Noriko; Koda, Toshiaki; Kakinuma, Mitsuaki; Nishimura, Susumu

1989-01-01

Synthetic human c‐Ha‐ras genes in which amino acid codons were altered to those which are frequently used in highly expressed Escherichia coli genes were ligated to the 3′‐end of Rous sarcoma virus long terminal repeat. When NIH3T3 cells were transfected with the plasmids having those genes with valine at codon 12, leucine at codon 61 or arginine at codon 61, transformants were efficiently produced. These results indicated that the synthetic c‐Ha‐ras genes are expressed in a mammalian system even though their codon usage is altered to correspond with that of E. colt. This expression vector system should he useful for studies on the structure‐function relationships of c‐Ha‐ras, since the synthetic gene can be easily modified to have multiple base alterations, and can also be used simultaneously for the production of large amounts of p21 in E. coli for biochemical and biophysical studies. PMID:2542206

RNA Editing in Plant Mitochondria

NASA Astrophysics Data System (ADS)

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

1989-12-01

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

An analysis of the metabolic theory of the origin of the genetic code

NASA Technical Reports Server (NTRS)

Amirnovin, R.; Bada, J. L. (Principal Investigator)

1997-01-01

A computer program was used to test Wong's coevolution theory of the genetic code. The codon correlations between the codons of biosynthetically related amino acids in the universal genetic code and in randomly generated genetic codes were compared. It was determined that many codon correlations are also present within random genetic codes and that among the random codes there are always several which have many more correlations than that found in the universal code. Although the number of correlations depends on the choice of biosynthetically related amino acids, the probability of choosing a random genetic code with the same or greater number of codon correlations as the universal genetic code was found to vary from 0.1% to 34% (with respect to a fairly complete listing of related amino acids). Thus, Wong's theory that the genetic code arose by coevolution with the biosynthetic pathways of amino acids, based on codon correlations between biosynthetically related amino acids, is statistical in nature.

Complete mitochondrial genome of the Yellownose skate: Zearaja chilensis (Rajiformes, Rajidae).

PubMed

Jeong, Dageum; Lee, Youn-Ho

2016-01-01

The complete sequence of mitochondrial DNA of a Yellownose skate, Zearaja chilensis was determined for the first time. It is 16,909 bp in length covering 2 rRNA, 22 tRNA and 13 protein coding genes with the identical gene order and structure as those of other Rajidae species. The nucleotide of L-strand is composed of low G (14.3%), and slightly high A + T (58.9%) nucleotides. The strong codon usage bias against the use of G (6.0%) is found at the third codon positions. Twelve of the 13 protein coding genes use ATG as the start codon while COX1 starts with GTG. As for the stop codon, only ND4 shows an incomplete stop codon TA. This is the first report of the mitogenome for a species in the genus Zearaja, providing a valuable source of genetic information on the evolution of the family Rajidae and the genus Zearaja as well as for establishment of a sustainble fishery management plan of the species.

Comprehensive analysis of the codon usage patterns in the envelope glycoprotein E2 gene of the classical swine fever virus

PubMed Central

Chi, Xiaojuan; Wang, Song; Ma, Yanmei; Chen, Jilong

2017-01-01

The classical swine fever virus (CSFV), circulating worldwide, is a highly contagious virus. Since the emergence of CSFV, it has caused great economic loss in swine industry. The envelope glycoprotein E2 gene of the CSFV is an immunoprotective antigen that induces the immune system to produce neutralizing antibodies. Therefore, it is essential to study the codon usage of the E2 gene of the CSFV. In this study, 140 coding sequences of the E2 gene were analyzed. The value of effective number of codons (ENC) showed low codon usage bias in the E2 gene. Our study showed that codon usage could be described mainly by mutation pressure ENC plot analysis combined with principal component analysis (PCA) and translational selection-correlation analysis between the general average hydropathicity (Gravy) and aromaticity (Aroma), and nucleotides at the third position of codons (A3s, T3s, G3s, C3s and GC3s). Furthermore, the neutrality analysis, which explained the relationship between GC12s and GC3s, revealed that natural selection had a key role compared with mutational bias during the evolution of the E2 gene. These results lay a foundation for further research on the molecular evolution of CSFV. PMID:28880881

Comprehensive analysis of the codon usage patterns in the envelope glycoprotein E2 gene of the classical swine fever virus.

PubMed

Chen, Ye; Li, Xinxin; Chi, Xiaojuan; Wang, Song; Ma, Yanmei; Chen, Jilong

2017-01-01

The classical swine fever virus (CSFV), circulating worldwide, is a highly contagious virus. Since the emergence of CSFV, it has caused great economic loss in swine industry. The envelope glycoprotein E2 gene of the CSFV is an immunoprotective antigen that induces the immune system to produce neutralizing antibodies. Therefore, it is essential to study the codon usage of the E2 gene of the CSFV. In this study, 140 coding sequences of the E2 gene were analyzed. The value of effective number of codons (ENC) showed low codon usage bias in the E2 gene. Our study showed that codon usage could be described mainly by mutation pressure ENC plot analysis combined with principal component analysis (PCA) and translational selection-correlation analysis between the general average hydropathicity (Gravy) and aromaticity (Aroma), and nucleotides at the third position of codons (A3s, T3s, G3s, C3s and GC3s). Furthermore, the neutrality analysis, which explained the relationship between GC12s and GC3s, revealed that natural selection had a key role compared with mutational bias during the evolution of the E2 gene. These results lay a foundation for further research on the molecular evolution of CSFV.

rpoB gene mutations among Mycobacterium tuberculosis isolates from extrapulmonary sites.

PubMed

Khosravi, Azar Dokht; Meghdadi, Hossein; Ghadiri, Ata A; Alami, Ameneh; Sina, Amir Hossein; Mirsaeidi, Mehdi

2018-03-01

The aim of this study was to analyze mutations occurring in the rpoB gene of Mycobacterium tuberculosis (MTB) isolates from clinical samples of extrapulmonary tuberculosis (EPTB). Seventy formalin-fixed, paraffin-embedded samples and fresh tissue samples from confirmed EPTB cases were analyzed. Nested PCR based on the rpoB gene was performed on the extracted DNAs, combined with cloning and subsequent sequencing. Sixty-seven (95.7%) samples were positive for nester PCR. Sequence analysis of the 81 bp region of the rpoB gene demonstrated mutations in 41 (61.2%) of 67 sequenced samples. Several point mutations including deletion mutations at codons 510, 512, 513 and 515, with 45% and 51% of the mutations in codons 512 and 513 respectively were seen, along with 26% replacement mutations at codons 509, 513, 514, 518, 520, 524 and 531. The most common alteration was Gln → His, at codon 513, presented in 30 (75.6%) isolates. This study demonstrated sequence alterations in codon 513 of the 81 bp region of the rpoB gene as the most common mutation occurred in 75.6% of molecularly confirmed rifampin-resistant strains. In addition, simultaneous mutation at codons 512 and 513 was demonstrated in 34.3% of the isolates. © 2018 APMIS. Published by John Wiley & Sons Ltd.

Differential Reprogramming of Isogenic Colorectal Cancer Cells by Distinct Activating KRAS Mutations

PubMed Central

2015-01-01

Oncogenic mutations of Ras at codons 12, 13, or 61, that render the protein constitutively active, are found in ∼16% of all cancer cases. Among the three major Ras isoforms, KRAS is the most frequently mutated isoform in cancer. Each Ras isoform and tumor type displays a distinct pattern of codon-specific mutations. In colon cancer, KRAS is typically mutated at codon 12, but a significant fraction of patients have mutations at codon 13. Clinical data suggest different outcomes and responsiveness to treatment between these two groups. To investigate the differential effects upon cell status associated with KRAS mutations we performed a quantitative analysis of the proteome and phosphoproteome of isogenic SW48 colon cancer cell lines in which one allele of the endogenous gene has been edited to harbor specific KRAS mutations (G12V, G12D, or G13D). Each mutation generates a distinct signature, with the most variability seen between G13D and the codon 12 KRAS mutants. One notable example of specific up-regulation in KRAS codon 12 mutant SW48 cells is provided by the short form of the colon cancer stem cell marker doublecortin-like Kinase 1 (DCLK1) that can be reversed by suppression of KRAS. PMID:25599653

Modification of orthogonal tRNAs: unexpected consequences for sense codon reassignment.

PubMed

Biddle, Wil; Schmitt, Margaret A; Fisk, John D

2016-12-01

Breaking the degeneracy of the genetic code via sense codon reassignment has emerged as a way to incorporate multiple copies of multiple non-canonical amino acids into a protein of interest. Here, we report the modification of a normally orthogonal tRNA by a host enzyme and show that this adventitious modification has a direct impact on the activity of the orthogonal tRNA in translation. We observed nearly equal decoding of both histidine codons, CAU and CAC, by an engineered orthogonal M. jannaschii tRNA with an AUG anticodon: tRNA Opt We suspected a modification of the tRNA Opt AUG anticodon was responsible for the anomalous lack of codon discrimination and demonstrate that adenosine 34 of tRNA Opt AUG is converted to inosine. We identified tRNA Opt AUG anticodon loop variants that increase reassignment of the histidine CAU codon, decrease incorporation in response to the histidine CAC codon, and improve cell health and growth profiles. Recognizing tRNA modification as both a potential pitfall and avenue of directed alteration will be important as the field of genetic code engineering continues to infiltrate the genetic codes of diverse organisms. © The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.

Cloning and expression of codon-optimized recombinant darbepoetin alfa in Leishmania tarentolae T7-TR.

PubMed

Kianmehr, Anvarsadat; Golavar, Raziyeh; Rouintan, Mandana; Mahrooz, Abdolkarim; Fard-Esfahani, Pezhman; Oladnabi, Morteza; Khajeniazi, Safoura; Mostafavi, Seyede Samaneh; Omidinia, Eskandar

2016-02-01

Darbepoetin alfa is an engineered and hyperglycosylated analog of recombinant human erythropoietin (EPO) which is used as a drug in treating anemia in patients with chronic kidney failure and cancer. This study desribes the secretory expression of a codon-optimized recombinant form of darbepoetin alfa in Leishmania tarentolae T7-TR. Synthetic codon-optimized gene was amplified by PCR and cloned into the pLEXSY-I-blecherry3 vector. The resultant expression vector, pLEXSYDarbo, was purified, digested, and electroporated into the L. tarentolae. Expression of recombinant darbepoetin alfa was evaluated by ELISA, reverse-transcription PCR (RT-PCR), Western blotting, and biological activity. After codon optimization, codon adaptation index (CAI) of the gene raised from 0.50 to 0.99 and its GC% content changed from 56% to 58%. Expression analysis confirmed the presence of a protein band at 40 kDa. Furthermore, reticulocyte experiment results revealed that the activity of expressed darbepoetin alfa was similar to that of its equivalent expressed in Chinese hamster ovary (CHO) cells. These data suggested that the codon optimization and expression in L. tarentolae host provided an efficient approach for high level expression of darbepoetin alfa. Copyright © 2015 Elsevier Inc. All rights reserved.

A dose rate causes no fluctuating asymmetry indexes changes in silver birch (Betula pendula (L.) Roth.) leaves and Scots pine (Pinus sylvestris L.) needles in the Chernobyl Exclusion Zone.

PubMed

Kashparova, Elena; Levchuk, Sviatoslav; Morozova, Valeriia; Kashparov, Valery

2018-06-04

The assessment of the fluctuating asymmetry based on measurement of the parameters of left and right parts of silver birch (Betula pendula (L.) Roth.) leaves and relative sizes of pairs of Scots pine (Pinus sylvestris L.) needles from the Chernobyl Exclusion Zone (ChEZ) was carried out. Twelve samples of both birch leaves and pairs of needles were collected from 10 trees at 5 sites in the Chernobyl Exclusion Zone and also at one control site located outside the ChEZ. Values of gamma dose rate in the air varied between the sites from 0.1 to 40 μGy h -1 . Activity concentrations of 90 Sr and 137 Cs in the birch leaves varied over the range of 0.9÷2460 kBq kg -1 and 0.1÷339 kBq·kg -1 (DW), respectively. In addition to the above, in the Scots pine needles, these ranges were 0.7 ÷1970 kBq kg -1 f for 90 Sr and 0.1÷78 kBq kg -1 (DW) for 137 Cs. From the values of the radionuclides activity concentrations in the plants, the internal dose rate is estimated to be in the range of 0.1 ÷ 274 μGy h -1 . The main sources of the internal dose rate were radiation of 90 Sr and 90 Y. Indices of fluctuating asymmetry of silver birch leaves and Scots pine needles varied over the range of 0.048 ± 0.007 ÷ 0.060 ± 0.009 and 0.014 ± 0.002 ÷ 0.018 ± 0.002, respectively, and did not statistically differ for all experimental sites. The indices also did not depend on the external or internal dose rate of ionizing radiation for plants. The above findings seem to be consistent with other research effort in terms of understanding the response of organisms to chronic pollutant exposure and the long-term effects of large scale nuclear accidents. Copyright © 2018 Elsevier Ltd. All rights reserved.

Historical changes in lead concentrations in tree-rings of sycamore, oak and Scots pine in north-west England.

PubMed

Watmough, Shaun A; Hutchinson, Thomas C

2002-07-03

Lead concentrations in tree rings of sycamore (Acer pseudoplatanus L.), oak (Quercus robur L.) and Scots pine (Pinus sylvestris L.) sampled at a parkland in north-west England were measured in wood formed since the mid-1800s. Concentrations of Pb in Scots pine and oak peaked in wood formed between 1900 and 1940, most likely because of Pb accumulation in heartwood, indicating that oak and Scots pine are unsuitable for monitoring temporal changes in Pb deposition at the study site. In contrast, Pb concentrations in sycamore, a species that has similar heartwood and sapwood chemistry, were relatively constant in wood formed between the mid-1800s and 1950. Lead concentrations decreased steadily in sycamore tree rings formed after the 1950s, and decreased more abruptly in wood formed after 1985. This sharp decrease in wood Pb cannot be due to decreases in soil Pb concentration. Stable Pb isotope analysis was used to further investigate Pb patterns in sycamore wood. Excess 206Pb/207Pb ratios in tree-rings of sycamore were relatively constant, approximately 1.17, in wood formed prior to the 1930s, but decreased steadily thereafter reaching a minimum value of approximately 1.16 in wood formed between 1975 and 1985 after which time 206Pb/207Pb ratios increased. This pattern is consistent with changes in Pb isotope ratios measured in peat, sediment and aerosol samples in the UK. However, the magnitude of the decrease in 206Pb/207Pb (largely due to gasoline Pb) is considerably lower than in other studies and our estimates indicate that less than 20% of the total Pb in sycamore wood measured since the mid-1800s is derived from gasoline emissions. A more likely explanation for the pattern of Pb observed in sycamore tree rings is that soil Pb accumulates within rings of the diffuse porous wood over a number of years. Such uptake patterns would result in lower Pb concentrations in the outer (more recently formed) tree rings, which coincide with recent reductions in Pb deposition in the UK. Overall, this study indicates that tree ring chemistry is unsuitable for monitoring historical changes in Pb deposition at the study site.

Surface Fire Influence on Carbon Balance Components in Scots Pine Forest of Siberia, Russia

NASA Astrophysics Data System (ADS)

Kukavskaya, E.; Ivanova, G. A.; Conard, S. G.; Soja, A. J.

2008-12-01

Wildfire is one of the most important disturbances in boreal forests, and it can have a profound effect on forest-atmosphere carbon exchange. Pinus sylvestris (Scots pine) stands of Siberia are strongly impacted by fires of low to high severity. Biomass distribution in mature lichen/feathermoss Scots pine stands indicates that they are carbon sinks before fire. Fires contribute significantly to the carbon budget resulting in a considerable carbon efflux, initially through direct consumption of forest fuels and later as a result of tree mortality and decomposition of dead material accumulated on the forest floor. In initial postfire years these processes dominate over photosynthetic carbon assimilation, and the ecosystems become a carbon source. Over several postfire years, above-ground carbon in dead biomass tends to increase, with the increase depending significantly on fire severity. High-severity fire enhances dead biomass carbon, while moderate- and low-severity fires have minimal effect on above-ground carbon distribution in Scots pine ecosystems. Dead stand biomass carbon increases, primarily during the first two years following fires, due to tree mortality. This increase can account for up to 12.4% of the total stand biomass after low- and moderate- intensity fires. We found tree dieback following a high-intensity fire is an order of magnitude higher, and thus the dead biomass increases up to 88.1% of total above-ground biomass. Photosynthetic CO2 uptake decreases with increasing tree mortality, and needle foliage and bark are incorporated into the upper layer of the forest floor in the course of years. Ground vegetation and duff carbon were >90, 71-83, and 82% of prefire levels after fires of low, moderate, and high severity, respectively for the first 4 to 5 years after fire. Fires of low and moderate severity caused down woody fuel carbon to increase by 2.1 and 3.6 t ha-1 respectively by four years after burning as compared to the pre-fire values. Climate change and increasing drought length observed in recent decades have increased the probability of high-intensity fire occurrence. Areas burned have increased in extent and severity across Siberia, resulting in increased carbon emissions to the atmosphere from fuel combustion and post fire decomposition.

Effect of KRAS codon13 mutations in patients with advanced colorectal cancer (advanced CRC) under oxaliplatin containing chemotherapy. Results from a translational study of the AIO colorectal study group

PubMed Central

2012-01-01

Background To evaluate the value of KRAS codon 13 mutations in patients with advanced colorectal cancer (advanced CRC) treated with oxaliplatin and fluoropyrimidines. Methods Tumor specimens from 201 patients with advanced CRC from a randomized, phase III trial comparing oxaliplatin/5-FU vs. oxaliplatin/capecitabine were retrospectively analyzed for KRAS mutations. Mutation data were correlated to response data (Overall response rate, ORR), progression-free survival (PFS) and overall survival (OS). Results 201 patients were analysed for KRAS mutation (61.2% males; mean age 64.2 ± 8.6 years). KRAS mutations were identified in 36.3% of tumors (28.8% in codon 12, 7.4% in codon 13). The ORR in codon 13 patients compared to codon 12 and wild type patients was significantly lower (p = 0.008). There was a tendency for a better overall survival in KRAS wild type patients compared to mutants (p = 0.085). PFS in all patients was not different in the three KRAS genetic groups (p = 0.72). However, we found a marked difference in PFS between patients with codon 12 and 13 mutant tumors treated with infusional 5-FU versus capecitabine based regimens. Conclusions Our data suggest that the type of KRAS mutation may be of clinical relevance under oxaliplatin combination chemotherapies without the addition of monoclonal antibodies in particular when overall response rates are important. Trial registration number 2002-04-017 PMID:22876876

Mitochondrial genetic codes evolve to match amino acid requirements of proteins.

PubMed

Swire, Jonathan; Judson, Olivia P; Burt, Austin

2005-01-01

Mitochondria often use genetic codes different from the standard genetic code. Now that many mitochondrial genomes have been sequenced, these variant codes provide the first opportunity to examine empirically the processes that produce new genetic codes. The key question is: Are codon reassignments the sole result of mutation and genetic drift? Or are they the result of natural selection? Here we present an analysis of 24 phylogenetically independent codon reassignments in mitochondria. Although the mutation-drift hypothesis can explain reassignments from stop to an amino acid, we found that it cannot explain reassignments from one amino acid to another. In particular--and contrary to the predictions of the mutation-drift hypothesis--the codon involved in such a reassignment was not rare in the ancestral genome. Instead, such reassignments appear to take place while the codon is in use at an appreciable frequency. Moreover, the comparison of inferred amino acid usage in the ancestral genome with the neutral expectation shows that the amino acid gaining the codon was selectively favored over the amino acid losing the codon. These results are consistent with a simple model of weak selection on the amino acid composition of proteins in which codon reassignments are selected because they compensate for multiple slightly deleterious mutations throughout the mitochondrial genome. We propose that the selection pressure is for reduced protein synthesis cost: most reassignments give amino acids that are less expensive to synthesize. Taken together, our results strongly suggest that mitochondrial genetic codes evolve to match the amino acid requirements of proteins.

On the Evolution of the Standard Genetic Code: Vestiges of Critical Scale Invariance from the RNA World in Current Prokaryote Genomes

PubMed Central

José, Marco V.; Govezensky, Tzipe; García, José A.; Bobadilla, Juan R.

2009-01-01

Herein two genetic codes from which the primeval RNA code could have originated the standard genetic code (SGC) are derived. One of them, called extended RNA code type I, consists of all codons of the type RNY (purine-any base-pyrimidine) plus codons obtained by considering the RNA code but in the second (NYR type) and third (YRN type) reading frames. The extended RNA code type II, comprises all codons of the type RNY plus codons that arise from transversions of the RNA code in the first (YNY type) and third (RNR) nucleotide bases. In order to test if putative nucleotide sequences in the RNA World and in both extended RNA codes, share the same scaling and statistical properties to those encountered in current prokaryotes, we used the genomes of four Eubacteria and three Archaeas. For each prokaryote, we obtained their respective genomes obeying the RNA code or the extended RNA codes types I and II. In each case, we estimated the scaling properties of triplet sequences via a renormalization group approach, and we calculated the frequency distributions of distances for each codon. Remarkably, the scaling properties of the distance series of some codons from the RNA code and most codons from both extended RNA codes turned out to be identical or very close to the scaling properties of codons of the SGC. To test for the robustness of these results, we show, via computer simulation experiments, that random mutations of current genomes, at the rates of 10−10 per site per year during three billions of years, were not enough for destroying the observed patterns. Therefore, we conclude that most current prokaryotes may still contain relics of the primeval RNA World and that both extended RNA codes may well represent two plausible evolutionary paths between the RNA code and the current SGC. PMID:19183813

Global analysis of translation termination in E. coli

PubMed Central

Baggett, Natalie E.

2017-01-01

Terminating protein translation accurately and efficiently is critical for both protein fidelity and ribosome recycling for continued translation. The three bacterial release factors (RFs) play key roles: RF1 and 2 recognize stop codons and terminate translation; and RF3 promotes disassociation of bound release factors. Probing release factors mutations with reporter constructs containing programmed frameshifting sequences or premature stop codons had revealed a propensity for readthrough or frameshifting at these specific sites, but their effects on translation genome-wide have not been examined. We performed ribosome profiling on a set of isogenic strains with well-characterized release factor mutations to determine how they alter translation globally. Consistent with their known defects, strains with increasingly severe release factor defects exhibit increasingly severe accumulation of ribosomes over stop codons, indicative of an increased duration of the termination/release phase of translation. Release factor mutant strains also exhibit increased occupancy in the region following the stop codon at a significant number of genes. Our global analysis revealed that, as expected, translation termination is generally efficient and accurate, but that at a significant number of genes (≥ 50) the ribosome signature after the stop codon is suggestive of translation past the stop codon. Even native E. coli K-12 exhibits the ribosome signature suggestive of protein extension, especially at UGA codons, which rely exclusively on the reduced function RF2 variant of the K-12 strain for termination. Deletion of RF3 increases the severity of the defect. We unambiguously demonstrate readthrough and frameshifting protein extensions and their further accumulation in mutant strains for a few select cases. In addition to enhancing recoding, ribosome accumulation over stop codons disrupts attenuation control of biosynthetic operons, and may alter expression of some overlapping genes. Together, these functional alterations may either augment the protein repertoire or produce deleterious proteins. PMID:28301469

Modifications modulate anticodon loop dynamics and codon recognition of E. coli tRNA(Arg1,2).

PubMed

Cantara, William A; Bilbille, Yann; Kim, Jia; Kaiser, Rob; Leszczyńska, Grażyna; Malkiewicz, Andrzej; Agris, Paul F

2012-03-02

Three of six arginine codons are read by two tRNA(Arg) isoacceptors in Escherichia coli. The anticodon stem and loop of these isoacceptors (ASL(Arg1,2)) differs only in that the position 32 cytidine of tRNA(Arg1) is posttranscriptionally modified to 2-thiocytidine (s(2)C(32)). The tRNA(Arg1,2) are also modified at positions 34 (inosine, I(34)) and 37 (2-methyladenosine, m(2)A(37)). To investigate the roles of modifications in the structure and function, we analyzed six ASL(Arg1,2) constructs differing in their array of modifications by spectroscopy and codon binding assays. Thermal denaturation and circular dichroism spectroscopy indicated that modifications contribute thermodynamic and base stacking properties, resulting in more order but less stability. NMR-derived structures of the ASL(Arg1,2) showed that the solution structures of the ASLs were nearly identical. Surprisingly, none possessed the U-turn conformation required for effective codon binding on the ribosome. Yet, all ASL(Arg1,2) constructs efficiently bound the cognate CGU codon. Three ASLs with I(34) were able to decode CGC, whereas only the singly modified ASL(Arg1,2)(ICG) with I(34) was able to decode CGA. The dissociation constants for all codon bindings were physiologically relevant (0.4-1.4 μM). However, with the introduction of s(2)C(32) or m(2)A(37) to ASL(Arg1,2)(ICG), the maximum amount of ASL bound to CGU and CGC was significantly reduced. These results suggest that, by allowing loop flexibility, the modifications modulate the conformation of the ASL(Arg1,2), which takes one structure free in solution and two others when bound to the cognate arginyl-tRNA synthetase or to codons on the ribosome where modifications reduce or restrict binding to specific codons. Copyright © 2011 Elsevier Ltd. All rights reserved.

Genetic and codon usage bias analyses of polymerase genes of equine influenza virus and its relation to evolution.

PubMed

Bera, Bidhan Ch; Virmani, Nitin; Kumar, Naveen; Anand, Taruna; Pavulraj, S; Rash, Adam; Elton, Debra; Rash, Nicola; Bhatia, Sandeep; Sood, Richa; Singh, Raj Kumar; Tripathi, Bhupendra Nath

2017-08-23

Equine influenza is a major health problem of equines worldwide. The polymerase genes of influenza virus have key roles in virus replication, transcription, transmission between hosts and pathogenesis. Hence, the comprehensive genetic and codon usage bias of polymerase genes of equine influenza virus (EIV) were analyzed to elucidate the genetic and evolutionary relationships in a novel perspective. The group - specific consensus amino acid substitutions were identified in all polymerase genes of EIVs that led to divergence of EIVs into various clades. The consistent amino acid changes were also detected in the Florida clade 2 EIVs circulating in Europe and Asia since 2007. To study the codon usage patterns, a total of 281,324 codons of polymerase genes of EIV H3N8 isolates from 1963 to 2015 were systemically analyzed. The polymerase genes of EIVs exhibit a weak codon usage bias. The ENc-GC3s and Neutrality plots indicated that natural selection is the major influencing factor of codon usage bias, and that the impact of mutation pressure is comparatively minor. The methods for estimating host imposed translation pressure suggested that the polymerase acidic (PA) gene seems to be under less translational pressure compared to polymerase basic 1 (PB1) and polymerase basic 2 (PB2) genes. The multivariate statistical analysis of polymerase genes divided EIVs into four evolutionary diverged clusters - Pre-divergent, Eurasian, Florida sub-lineage 1 and 2. Various lineage specific amino acid substitutions observed in all polymerase genes of EIVs and especially, clade 2 EIVs underwent major variations which led to the emergence of a phylogenetically distinct group of EIVs originating from Richmond/1/07. The codon usage bias was low in all the polymerase genes of EIVs that was influenced by the multiple factors such as the nucleotide compositions, mutation pressure, aromaticity and hydropathicity. However, natural selection was the major influencing factor in defining the codon usage patterns and evolution of polymerase genes of EIVs.

Codon 13 KRAS mutation predicts patterns of recurrence in patients undergoing hepatectomy for colorectal liver metastases.

PubMed

Margonis, Georgios A; Kim, Yuhree; Sasaki, Kazunari; Samaha, Mario; Amini, Neda; Pawlik, Timothy M

2016-09-01

Investigations regarding the impact of tumor biology after surgical management of colorectal liver metastasis have focused largely on overall survival. We investigated the impact of codon-specific KRAS mutations on the rates and patterns of recurrence in patients after surgery for colorectal liver metastasis (CRLM). All patients who underwent curative-intent surgery for CRLM between 2002 and 2015 at Johns Hopkins who had available data on KRAS mutation status were identified. Clinico-pathologic data, recurrence patterns, and recurrence-free survival (RFS) were assessed using univariable and multivariable analyses. A total of 512 patients underwent resection only (83.2%) or resection plus radiofrequency ablation (16.8%). Although 5-year overall survival was 64.6%, 284 (55.5%) patients recurred with a median RFS time of 18.1 months. The liver was the initial recurrence site for 181 patients, whereas extrahepatic recurrence was observed in 162 patients. Among patients with an extrahepatic recurrence, 102 (63%) had a lung recurrence. Although overall KRAS mutation was not associated with overall RFS (P = 0.186), it was independently associated with a worse extrahepatic (P = 0.004) and lung RFS (P = 0.007). Among patients with known KRAS codon-specific mutations, patients with codon 13 KRAS mutation had a worse 5-year extrahepatic RFS (P = 0.01), whereas codon 12 mutations were not associated with extrahepatic (P = 0.11) or lung-specific recurrence rate (P = 0.24). On multivariable analysis, only codon 13 mutation independently predicted worse overall extrahepatic RFS (P = 0.004) and lung-specific RFS (P = 0.023). Among patients undergoing resection of CRLM, overall KRAS mutation was not associated with RFS. KRAS codon 13 mutations, but not codon 12 mutations, were associated with a higher risk for overall extrahepatic recurrence and lung-specific recurrence. Cancer 2016. © 2016 American Cancer Society. Cancer 2016;122:2698-2707. © 2016 American Cancer Society. © 2016 American Cancer Society.

Simultaneous identification of 36 mutations in KRAS codons 61and 146, BRAF, NRAS, and PIK3CA in a single reaction by multiplex assay kit

PubMed Central

2013-01-01

Background Retrospective analyses in the West suggest that mutations in KRAS codons 61 and 146, BRAF, NRAS, and PIK3CA are negative predictive factors for cetuximab treatment in colorectal cancer patients. We developed a novel multiplex kit detecting 36 mutations in KRAS codons 61 and 146, BRAF, NRAS, and PIK3CA using Luminex (xMAP) assay in a single reaction. Methods Tumor samples and clinical data from Asian colorectal cancer patients treated with cetuximab were collected. We investigated KRAS, BRAF, NRAS, and PIK3CA mutations using both the multiplex kit and direct sequencing methods, and evaluated the concordance between the 2 methods. Objective response, progression-free survival (PFS), and overall survival (OS) were also evaluated according to mutational status. Results In total, 82 of 83 samples (78 surgically resected specimens and 5 biopsy specimens) were analyzed using both methods. All multiplex assays were performed using 50 ng of template DNA. The concordance rate between the methods was 100%. Overall, 49 (59.8%) patients had all wild-type tumors, 21 (25.6%) had tumors harboring KRAS codon 12 or 13 mutations, and 12 (14.6%) had tumors harboring KRAS codon 61, KRAS codon 146, BRAF, NRAS, or PIK3CA mutations. The response rates in these patient groups were 38.8%, 4.8%, and 0%, respectively. Median PFS in these groups was 6.1 months (95% confidence interval (CI): 3.1–9.2), 2.7 months (1.2–4.2), and 1.6 months (1.5–1.7); median OS was 13.8 months (9.2–18.4), 8.2 months (5.7–10.7), and 6.3 months (1.3–11.3), respectively. Statistically significant differences in both PFS and OS were found between patients with all wild-type tumors and those with KRAS codon 61, KRAS codon 146, BRAF, NRAS, or PIK3CA mutations (PFS: 95% CI, 0.11–0.44; P < 0.0001; OS: 95% CI, 0.15–0.61; P < 0.0001). Conclusions Our newly developed multiplex kit is practical and feasible for investigation of a range of sample types. Moreover, mutations in KRAS codon 61, KRAS codon 146, BRAF, NRAS, or PIK3CA detected in Asian patients were not predictive of clinical benefits from cetuximab treatment, similar to the result obtained in European studies. PMID:24006859

Role of a Novel I1781T Mutation and Other Mechanisms in Conferring Resistance to Acetyl-CoA Carboxylase Inhibiting Herbicides in a Black-Grass Population

PubMed Central

Kaundun, Shiv Shankhar; Hutchings, Sarah-Jane; Dale, Richard P.; McIndoe, Eddie

2013-01-01

Background Knowledge of the mechanisms of herbicide resistance is important for designing long term sustainable weed management strategies. Here, we have used an integrated biology and molecular approach to investigate the mechanisms of resistance to acetyl-CoA carboxylase inhibiting herbicides in a UK black-grass population (BG2). Methodology/Principal Findings Comparison between BG2 phenotypes using single discriminant rates of herbicides and genotypes based on ACCase gene sequencing showed that the I1781L, a novel I1781T, but not the W2027C mutations, were associated with resistance to cycloxydim. All plants were killed with clethodim and a few individuals containing the I1781L mutation were partially resistant to tepraloxydim. Whole plant dose response assays demonstrated that a single copy of the mutant T1781 allele conferred fourfold resistance levels to cycloxydim and clodinafop-propargyl. In contrast, the impact of the I1781T mutation was low (Rf = 1.6) and non-significant on pinoxaden. BG2 was also characterised by high levels of resistance, very likely non-target site based, to the two cereal selective herbicides clodinafop-propargyl and pinoxaden and not to the poorly metabolisable cyclohexanedione herbicides. Analysis of 480 plants from 40 cycloxydim resistant black grass populations from the UK using two very effective and high throughput dCAPS assays established for detecting any amino acid changes at the 1781 ACCase codon and for positively identifying the threonine residue, showed that the occurrence of the T1781 is extremely rare compared to the L1781 allele. Conclusion/Significance This study revealed a novel mutation at ACCase codon position 1781 and adequately assessed target site and non-target site mechanisms in conferring resistance to several ACCase herbicides in a black-grass population. It highlights that over time the level of suspected non-target site resistance to some cereal selective ACCase herbicides have in some instances surpassed that of target site resistance, including the one endowed by the most commonly encountered I1781L mutation. PMID:23936046

Peptide Conjugated Phosphorodiamidate Morpholino Oligomers Increase Survival of Mice Challenged with Ames Bacillus anthracis

PubMed Central

Geller, Bruce L.; Mellbye, Brett; Lane, Douglas; Iversen, Patrick L.; Bavari, Sina

2012-01-01

Targeting bacterial essential genes using antisense phosphorodiamidate morpholino oligomers (PMOs) represents an important strategy in the development of novel antibacterial therapeutics. PMOs are neutral DNA analogues that inhibit gene expression in a sequence-specific manner. In this study, several cationic, membrane-penetrating peptides were conjugated to PMOs (PPMOs) that target 2 bacterial essential genes: acyl carrier protein (acpP) and gyrase A (gyrA). These were tested for their ability to inhibit growth of Bacillus anthracis, a gram-positive spore-forming bacterium and causative agent of anthrax. PPMOs targeted upstream of both target gene start codons and conjugated with the bacterium-permeating peptide (RFF)3R were found to be most effective in inhibiting bacterial growth in vitro. Both of the gene-targeted PPMOs protected macrophages from B. anthracis induced cell death. Subsequent, in vivo testing of the PPMOs resulted in increased survival of mice challenged with the virulent Ames strain of B. anthracis. Together, these studies suggest that PPMOs targeting essential genes have the potential of being used as antisense antibiotics to treat B. anthracis infections. PMID:22978365

Abundant off-target edits from site-directed RNA editing can be reduced by nuclear localization of the editing enzyme.

PubMed

Vallecillo-Viejo, Isabel C; Liscovitch-Brauer, Noa; Montiel-Gonzalez, Maria Fernanda; Eisenberg, Eli; Rosenthal, Joshua J C

2018-01-02

Site-directed RNA editing (SDRE) is a general strategy for making targeted base changes in RNA molecules. Although the approach is relatively new, several groups, including our own, have been working on its development. The basic strategy has been to couple the catalytic domain of an adenosine (A) to inosine (I) RNA editing enzyme to a guide RNA that is used for targeting. Although highly efficient on-target editing has been reported, off-target events have not been rigorously quantified. In this report we target premature termination codons (PTCs) in messages encoding both a fluorescent reporter protein and the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) protein transiently transfected into human epithelial cells. We demonstrate that while on-target editing is efficient, off-target editing is extensive, both within the targeted message and across the entire transcriptome of the transfected cells. By redirecting the editing enzymes from the cytoplasm to the nucleus, off-target editing is reduced without compromising the on-target editing efficiency. The addition of the E488Q mutation to the editing enzymes, a common strategy for increasing on-target editing efficiency, causes a tremendous increase in off-target editing. These results underscore the need to reduce promiscuity in current approaches to SDRE.

Analysis of amino acid and codon usage in Paramecium bursaria.

PubMed

Dohra, Hideo; Fujishima, Masahiro; Suzuki, Haruo

2015-10-07

The ciliate Paramecium bursaria harbors the green-alga Chlorella symbionts. We reassembled the P. bursaria transcriptome to minimize falsely fused transcripts, and investigated amino acid and codon usage using the transcriptome data. Surface proteins preferentially use smaller amino acid residues like cysteine. Unusual synonymous codon and amino acid usage in highly expressed genes can reflect a balance between translational selection and other factors. A correlation of gene expression level with synonymous codon or amino acid usage is emphasized in genes down-regulated in symbiont-bearing cells compared to symbiont-free cells. Our results imply that the selection is associated with P. bursaria-Chlorella symbiosis. Copyright © 2015 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

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

PubMed Central

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

2007-01-01

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

Polymorphism in xeroderma pigmentosum complementation group C codon 939 and aflatoxin B1-related hepatocellular carcinoma in the Guangxi population.

PubMed

Long, Xi-Dai; Ma, Yun; Zhou, Yuan-Feng; Ma, Ai-Min; Fu, Guo-Hui

2010-10-01

Genetic polymorphisms in DNA repair genes may influence individual variations in DNA repair capacity, and this may be associated with the risk and outcome of hepatocellular carcinoma (HCC) related to aflatoxin B1 (AFB1) exposure. In this study, we focused on the polymorphism of xeroderma pigmentosum complementation group C (XPC) codon 939 (rs#2228001), which is involved in nucleotide excision repair. We conducted a case-control study including 1156 HCC cases and 1402 controls without any evidence of hepatic disease to evaluate the associations between this polymorphism and HCC risk and prognosis in the Guangxi population. AFB1 DNA adduct levels, XPC genotypes, and XPC protein levels were tested with a comparative enzyme-linked immunosorbent assay, TaqMan polymerase chain reaction for XPC genotypes, and immunohistochemistry, respectively. Higher AFB1 exposure was observed among HCC patients versus the control group [odds ratio (OR) = 9.88 for AFB1 exposure years and OR = 6.58 for AFB1 exposure levels]. The XPC codon 939 Gln alleles significantly increased HCC risk [OR = 1.25 (95% confidence interval = 1.03-1.52) for heterozygotes of the XPC codon 939 Lys and Gln alleles (XPC-LG) and OR = 1.81 (95% confidence interval = 1.36-2.40) for homozygotes of the XPC codon 939 Gln alleles (XPC-GG)]. Significant interactive effects between genotypes and AFB1 exposure status were also observed in the joint-effects analysis. This polymorphism, moreover, was correlated with XPC expression levels in cancerous tissues (r = -0.369, P < 0.001) and with the overall survival of HCC patients (the median survival times were 30, 25, and 19 months for patients with homozygotes of the XPC codon 939 Lys alleles, XPC-LG, and XPC-GG, respectively), especially under high AFB1 exposure conditions. Like AFB1 exposure, the XPC codon 939 polymorphism was an independent prognostic factor influencing the survival of HCC. Additionally, this polymorphism multiplicatively interacted with the xeroderma pigmentosum complementation group D codon 751 polymorphism with respect to HCC risk (OR(interaction) = 1.71). These results suggest that the XPC codon 939 polymorphism may be associated with the risk and outcome of AFB1-related HCC in the Guangxi population and may interact with AFB1 exposure in the process of HCC induction by AFB1.

Molecular Characterization of β-Thalassemia Mutations in Central Vietnam.

PubMed

Doro, Maria G; Casu, Giuseppina; Frogheri, Laura; Persico, Ivana; Triet, Le Phan Minh; Hoa, Phan Thi Thuy; Hoang, Nguyen Huy; Pirastru, Monica; Mereu, Paolo; Cucca, Francesco; Masala, Bruno

2017-03-01

The molecular basis of β-thalassemia (β-thal) mutations in North and in South Vietnam have been described during the past 15 years, whereas limited data were available concerning the central area of the country. In this study, we describe the molecular characterization and frequency of β-globin gene mutations in the Thua Thien Hue Province of Central Vietnam as the result of a first survey conducted in 22 transfusion-dependent patients, and four unrelated heterozygotes. Nine different known mutations were identified (seven of the β 0 and two of the β + type) in a total of 48 chromosomes. The most common was codon 26 (G>A) or Hb E (HBB: c.79 G>A) accounting for 29.2% of the total studied chromosomes, followed by codon 17 (A>T) (HBB: c.52 A>T) (25.0%), and codons 41/42 (-TTCT) (HBB: c.126_129delCTTT) (18.8%). Other mutations with appreciable frequencies (6.3-8.3%) were IVS-I-1 (G>T) (HBB: c.92+1 G>T), codon 26 (G>T) (HBB: c.79 G>T) and codons 71/72 (+A) (HBB: c.216_217insA). Relatively rarer (2.0%) were the promoter -28 (A>G) (HBB: c.78 A>G) mutation, the codon 95 (+A) (HBB: c.287_288insA), which is reported only in the Vietnamese, and the codons 14/15 (+G) (HBB: c.45_46insG) mutation, thus far observed only in Thailand. Results are relevant for implementing appropriate measures for β-thal prevention and control in the region as well as in the whole country.

Hand gesture recognition by analysis of codons

NASA Astrophysics Data System (ADS)

Ramachandra, Poornima; Shrikhande, Neelima

2007-09-01

The problem of recognizing gestures from images using computers can be approached by closely understanding how the human brain tackles it. A full fledged gesture recognition system will substitute mouse and keyboards completely. Humans can recognize most gestures by looking at the characteristic external shape or the silhouette of the fingers. Many previous techniques to recognize gestures dealt with motion and geometric features of hands. In this thesis gestures are recognized by the Codon-list pattern extracted from the object contour. All edges of an image are described in terms of sequence of Codons. The Codons are defined in terms of the relationship between maxima, minima and zeros of curvature encountered as one traverses the boundary of the object. We have concentrated on a catalog of 24 gesture images from the American Sign Language alphabet (Letter J and Z are ignored as they are represented using motion) [2]. The query image given as an input to the system is analyzed and tested against the Codon-lists, which are shape descriptors for external parts of a hand gesture. We have used the Weighted Frequency Indexing Transform (WFIT) approach which is used in DNA sequence matching for matching the Codon-lists. The matching algorithm consists of two steps: 1) the query sequences are converted to short sequences and are assigned weights and, 2) all the sequences of query gestures are pruned into match and mismatch subsequences by the frequency indexing tree based on the weights of the subsequences. The Codon sequences with the most weight are used to determine the most precise match. Once a match is found, the identified gesture and corresponding interpretation are shown as output.

The impact of KRAS mutations on VEGF-A production and tumour vascular network

PubMed Central

2013-01-01

Background The malignant potential of tumour cells may be influenced by the molecular nature of KRAS mutations being codon 13 mutations less aggressive than codon 12 ones. Their metabolic profile is also different, with an increased anaerobic glycolytic metabolism in cells harbouring codon 12 KRAS mutations compared with cells containing codon 13 mutations. We hypothesized that this distinct metabolic behaviour could be associated with different HIF-1α expression and a distinct angiogenic profile. Methods Codon13 KRAS mutation (ASP13) or codon12 KRAS mutation (CYS12) NIH3T3 transfectants were analyzed in vitro and in vivo. Expression of HIF-1α, and VEGF-A was studied at RNA and protein levels. Regulation of VEGF-A promoter activity was assessed by means of luciferase assays using different plasmid constructs. Vascular network was assessed in tumors growing after subcutaneous inoculation. Non parametric statistics were used for analysis of results. Results Our results show that in normoxic conditions ASP13 transfectants exhibited less HIF-1α protein levels and activity than CYS12. In contrast, codon 13 transfectants exhibited higher VEGF-A mRNA and protein levels and enhanced VEGF-A promoter activity. These differences were due to a differential activation of Sp1/AP2 transcription elements of the VEGF-A promoter associated with increased ERKs signalling in ASP13 transfectants. Subcutaneous CYS12 tumours expressed less VEGF-A and showed a higher microvessel density (MVD) than ASP13 tumours. In contrast, prominent vessels were only observed in the latter. Conclusion Subtle changes in the molecular nature of KRAS oncogene activating mutations occurring in tumour cells have a major impact on the vascular strategy devised providing with new insights on the role of KRAS mutations on angiogenesis. PMID:23506169

Analysis of phylogeny and codon usage bias and relationship of GC content, amino acid composition with expression of the structural nif genes.

PubMed

Mondal, Sunil Kanti; Kundu, Sudip; Das, Rabindranath; Roy, Sujit

2016-08-01

Bacteria and archaea have evolved with the ability to fix atmospheric dinitrogen in the form of ammonia, catalyzed by the nitrogenase enzyme complex which comprises three structural genes nifK, nifD and nifH. The nifK and nifD encodes for the beta and alpha subunits, respectively, of component 1, while nifH encodes for component 2 of nitrogenase. Phylogeny based on nifDHK have indicated that Cyanobacteria is closer to Proteobacteria alpha and gamma but not supported by the tree based on 16SrRNA. The evolutionary ancestor for the different trees was also different. The GC1 and GC2% analysis showed more consistency than GC3% which appeared to below for Firmicutes, Cyanobacteria and Euarchaeota while highest in Proteobacteria beta and clearly showed the proportional effect on the codon usage with a few exceptions. Few genes from Firmicutes, Euryarchaeota, Proteobacteria alpha and delta were found under mutational pressure. These nif genes with low and high GC3% from different classes of organisms showed similar expected number of codons. Distribution of the genes and codons, based on codon usage demonstrated opposite pattern for different orientation of mirror plane when compared with each other. Overall our results provide a comprehensive analysis on the evolutionary relationship of the three structural nif genes, nifK, nifD and nifH, respectively, in the context of codon usage bias, GC content relationship and amino acid composition of the encoded proteins and exploration of crucial statistical method for the analysis of positive data with non-constant variance to identify the shape factors of codon adaptation index.

Negative and Translation Termination-Dependent Positive Control of FLI-1 Protein Synthesis by Conserved Overlapping 5′ Upstream Open Reading Frames in Fli-1 mRNA

PubMed Central

Sarrazin, Sandrine; Starck, Joëlle; Gonnet, Colette; Doubeikovski, Alexandre; Melet, Fabrice; Morle, François

2000-01-01

The proto-oncogene Fli-1 encodes a transcription factor of the ets family whose overexpression is associated with multiple virally induced leukemias in mouse, inhibits murine and avian erythroid cell differentiation, and induces drastic perturbations of early development in Xenopus. This study demonstrates the surprisingly sophisticated regulation of Fli-1 mRNA translation. We establish that two FLI-1 protein isoforms (of 51 and 48 kDa) detected by Western blotting in vivo are synthesized by alternative translation initiation through the use of two highly conserved in-frame initiation codons, AUG +1 and AUG +100. Furthermore, we show that the synthesis of these two FLI-1 isoforms is regulated by two short overlapping 5′ upstream open reading frames (uORF) beginning at two highly conserved upstream initiation codons, AUG −41 and GUG −37, and terminating at two highly conserved stop codons, UGA +35 and UAA +15. The mutational analysis of these two 5′ uORF revealed that each of them negatively regulates FLI-1 protein synthesis by precluding cap-dependent scanning to the 48- and 51-kDa AUG codons. Simultaneously, the translation termination of the two 5′ uORF appears to enhance 48-kDa protein synthesis, by allowing downstream reinitiation at the 48-kDa AUG codon, and 51-kDa protein synthesis, by allowing scanning ribosomes to pile up and consequently allowing upstream initiation at the 51-kDa AUG codon. To our knowledge, this is the first example of a cellular mRNA displaying overlapping 5′ uORF whose translation termination appears to be involved in the positive control of translation initiation at both downstream and upstream initiation codons. PMID:10757781

Genotyping of beta thalassemia trait by high-resolution DNA melting analysis.

PubMed

Saetung, Rattika; Ongchai, Siriwan; Charoenkwan, Pimlak; Sanguansermsri, Torpong

2013-11-01

Beta thalassemia is a common hereditary hemalogogical disease in Thailand, with a prevalence of 5-8%. In this study, we evaluated the high resolution DNA melting (HRM) assay to identify beta thalassemia mutation in samples from 143 carriers of the beta thalassemia traits in at risk couples. The DNA was isolated from venous blood samples and tested for mutation under a series of 5 PCR-HRM (A, B, C, D and E primers) protocols. The A primers were for detection of beta thalassemia mutations in the HBB promoter region, the B primers for mutations in exon I, the C primers for exon II, the D primers for exon III and the E primers for the 3.4 kb deletion mutation. The mutations were diagnosed by comparing the complete melting curve profiles of a wild type control with those for each mutant sample. With the PCR-HRM technique, fourteen types of beta thalassemia mutations were detected. Each mutation had a unique and specific melting profile. The mutations included 36.4% (52 cases) codon 41/42-CTTT, 26.6% (38 cases) codon 17 A-T, 11.2% (16 cases) IVS1-1 G-T, 8.4% (12 cases) codon 71/72 +A, 8.4% (12 cases) of the 3.4 kb deletion and 3.5% (5 cases) -28 A-G. The remainder included one instance each of -87 C-A, -31 A-C, codon 27/28 +C, codon 30 G-A, IVS1-5 G-C, codon 35 C-A, codon 41-C and IVSII -654 C-T. Of the total cases, 85.8% of the mutations could be detected by primers B and C. The PCR-HRM method provides a rapid, simple and highly feasible strategy for mutation screening of beta thalassemia traits.

An expanded genetic code in mammalian cells with a functional quadruplet codon.

PubMed

Niu, Wei; Schultz, Peter G; Guo, Jiantao

2013-07-19

We have utilized in vitro evolution to identify tRNA variants with significantly enhanced activity for the incorporation of unnatural amino acids into proteins in response to a quadruplet codon in both bacterial and mammalian cells. This approach will facilitate the creation of an optimized and standardized system for the genetic incorporation of unnatural amino acids using quadruplet codons, which will allow the biosynthesis of biopolymers that contain multiple unnatural building blocks.

Regions of extreme synonymous codon selection in mammalian genes

PubMed Central

Schattner, Peter; Diekhans, Mark

2006-01-01

Recently there has been increasing evidence that purifying selection occurs among synonymous codons in mammalian genes. This selection appears to be a consequence of either cis-regulatory motifs, such as exonic splicing enhancers (ESEs), or mRNA secondary structures, being superimposed on the coding sequence of the gene. We have developed a program to identify regions likely to be enriched for such motifs by searching for extended regions of extreme codon conservation between homologous genes of related species. Here we present the results of applying this approach to five mammalian species (human, chimpanzee, mouse, rat and dog). Even with very conservative selection criteria, we find over 200 regions of extreme codon conservation, ranging in length from 60 to 178 codons. The regions are often found within genes involved in DNA-binding, RNA-binding or zinc-ion-binding. They are highly depleted for synonymous single nucleotide polymorphisms (SNPs) but not for non-synonymous SNPs, further indicating that the observed codon conservation is being driven by negative selection. Forty-three percent of the regions overlap conserved alternative transcript isoforms and are enriched for known ESEs. Other regions are enriched for TpA dinucleotides and may contain conserved motifs/structures relating to mRNA stability and/or degradation. We anticipate that this tool will be useful for detecting regions enriched in other classes of coding-sequence motifs and structures as well. PMID:16556911

The effect of tRNA levels on decoding times of mRNA codons.

PubMed

Dana, Alexandra; Tuller, Tamir

2014-08-01

The possible effect of transfer ribonucleic acid (tRNA) concentrations on codons decoding time is a fundamental biomedical research question; however, due to a large number of variables affecting this process and the non-direct relation between them, a conclusive answer to this question has eluded so far researchers in the field. In this study, we perform a novel analysis of the ribosome profiling data of four organisms which enables ranking the decoding times of different codons while filtering translational phenomena such as experimental biases, extreme ribosomal pauses and ribosome traffic jams. Based on this filtering, we show for the first time that there is a significant correlation between tRNA concentrations and the codons estimated decoding time both in prokaryotes and in eukaryotes in natural conditions (-0.38 to -0.66, all P values <0.006); in addition, we show that when considering tRNA concentrations, codons decoding times are not correlated with aminoacyl-tRNA levels. The reported results support the conjecture that translation efficiency is directly influenced by the tRNA levels in the cell. Thus, they should help to understand the evolution of synonymous aspects of coding sequences via the adaptation of their codons to the tRNA pool. © The Author(s) 2014. Published by Oxford University Press on behalf of Nucleic Acids Research.

Genotype-specific signal generation based on digestion of 3-way DNA junctions: application to KRAS variation detection.

PubMed

Amicarelli, Giulia; Adlerstein, Daniel; Shehi, Erlet; Wang, Fengfei; Makrigiorgos, G Mike

2006-10-01

Genotyping methods that reveal single-nucleotide differences are useful for a wide range of applications. We used digestion of 3-way DNA junctions in a novel technology, OneCutEventAmplificatioN (OCEAN) that allows sequence-specific signal generation and amplification. We combined OCEAN with peptide-nucleic-acid (PNA)-based variant enrichment to detect and simultaneously genotype v-Ki-ras2 Kirsten rat sarcoma viral oncogene homolog (KRAS) codon 12 sequence variants in human tissue specimens. We analyzed KRAS codon 12 sequence variants in 106 lung cancer surgical specimens. We conducted a PNA-PCR reaction that suppresses wild-type KRAS amplification and genotyped the product with a set of OCEAN reactions carried out in fluorescence microplate format. The isothermal OCEAN assay enabled a 3-way DNA junction to form between the specific target nucleic acid, a fluorescently labeled "amplifier", and an "anchor". The amplifier-anchor contact contains the recognition site for a restriction enzyme. Digestion produces a cleaved amplifier and generation of a fluorescent signal. The cleaved amplifier dissociates from the 3-way DNA junction, allowing a new amplifier to bind and propagate the reaction. The system detected and genotyped KRAS sequence variants down to approximately 0.3% variant-to-wild-type alleles. PNA-PCR/OCEAN had a concordance rate with PNA-PCR/sequencing of 93% to 98%, depending on the exact implementation. Concordance rate with restriction endonuclease-mediated selective-PCR/sequencing was 89%. OCEAN is a practical and low-cost novel technology for sequence-specific signal generation. Reliable analysis of KRAS sequence alterations in human specimens circumvents the requirement for sequencing. Application is expected in genotyping KRAS codon 12 sequence variants in surgical specimens or in bodily fluids, as well as single-base variations and sequence alterations in other genes.

Bacterial genomes lacking long-range correlations may not be modeled by low-order Markov chains: the role of mixing statistics and frame shift of neighboring genes.

PubMed

Cocho, Germinal; Miramontes, Pedro; Mansilla, Ricardo; Li, Wentian

2014-12-01

We examine the relationship between exponential correlation functions and Markov models in a bacterial genome in detail. Despite the well known fact that Markov models generate sequences with correlation function that decays exponentially, simply constructed Markov models based on nearest-neighbor dimer (first-order), trimer (second-order), up to hexamer (fifth-order), and treating the DNA sequence as being homogeneous all fail to predict the value of exponential decay rate. Even reading-frame-specific Markov models (both first- and fifth-order) could not explain the fact that the exponential decay is very slow. Starting with the in-phase coding-DNA-sequence (CDS), we investigated correlation within a fixed-codon-position subsequence, and in artificially constructed sequences by packing CDSs with out-of-phase spacers, as well as altering CDS length distribution by imposing an upper limit. From these targeted analyses, we conclude that the correlation in the bacterial genomic sequence is mainly due to a mixing of heterogeneous statistics at different codon positions, and the decay of correlation is due to the possible out-of-phase between neighboring CDSs. There are also small contributions to the correlation from bases at the same codon position, as well as by non-coding sequences. These show that the seemingly simple exponential correlation functions in bacterial genome hide a complexity in correlation structure which is not suitable for a modeling by Markov chain in a homogeneous sequence. Other results include: use of the (absolute value) second largest eigenvalue to represent the 16 correlation functions and the prediction of a 10-11 base periodicity from the hexamer frequencies. Copyright © 2014 Elsevier Ltd. All rights reserved.

Co-amplification at lower denaturation-temperature PCR combined with unlabled-probe high-resolution melting to detect KRAS codon 12 and 13 mutations in plasma-circulating DNA of pancreatic adenocarcinoma cases.

PubMed

Wu, Jiong; Zhou, Yan; Zhang, Chun-Yan; Song, Bin-Bin; Wang, Bei-Li; Pan, Bai-Shen; Lou, Wen-Hui; Guo, Wei

2014-01-01

The aim of our study was to establish COLD-PCR combined with an unlabeled-probe HRM approach for detecting KRAS codon 12 and 13 mutations in plasma-circulating DNA of pancreatic adenocarcinoma (PA) cases as a novel and effective diagnostic technique. We tested the sensitivity and specificity of this approach with dilutions of known mutated cell lines. We screened 36 plasma-circulating DNA samples, 24 from the disease control group and 25 of a healthy group, to be subsequently sequenced to confirm mutations. Simultaneously, we tested the specimens using conventional PCR followed by HRM and then used target-DNA cloning and sequencing for verification. The ROC and respective AUC were calculated for KRAS mutations and/or serum CA 19-9. It was found that the sensitivity of Sanger reached 0.5% with COLD- PCR, whereas that obtained after conventional PCR did 20%; that of COLD-PCR based on unlabeled-probe HRM, 0.1%. KRAS mutations were identified in 26 of 36 PA cases (72.2%), while none were detected in the disease control and/or healthy group. KRAS mutations were identified both in 26 PA tissues and plasma samples. The AUC of COLD-PCR based unlabeled probe HRM turned out to be 0.861, which when combined with CA 19-9 increased to 0.934. It was concluded that COLD-PCR with unlabeled-probe HRM can be a sensitive and accurate screening technique to detect KRAS codon 12 and 13 mutations in plasma-circulating DNA for diagnosing and treating PA.

MicroRNA Targeting Specificity in Mammals: Determinants Beyond Seed Pairing

PubMed Central

Grimson, Andrew; Farh, Kyle Kai-How; Johnston, Wendy K.; Garrett-Engele, Philip; Lim, Lee P.; Bartel, David P.

2013-01-01

Summary Mammalian microRNAs (miRNAs) pair to 3'UTRs of mRNAs to direct their posttranscriptional repression. Important for target recognition are ~7-nt sites that match the seed region of the miRNA. However, these seed matches are not always sufficient for repression, indicating that other characteristics help specify targeting. By combining computational and experimental approaches, we uncovered five general features of site context that boost site efficacy: AU-rich nucleotide composition near the site, proximity to sites for co-expressed miRNAs (which leads to cooperative action), proximity to residues pairing to miRNA nucleotides 13–16, and positioning within the 3'UTR at least 15 nt from the stop codon and away from the center of long UTRs. A model combining these context determinants quantitatively predicts site performance both for exogenously added miRNAs and for endogenous miRNA-message interactions. Because it predicts site efficacy without recourse to evolutionary conservation, the model also identifies effective nonconserved sites and siRNA off-targets. PMID:17612493

Alignment-based and alignment-free methods converge with experimental data on amino acids coded by stop codons at split between nuclear and mitochondrial genetic codes.

PubMed

Seligmann, Hervé

2018-05-01

Genetic codes mainly evolve by reassigning punctuation codons, starts and stops. Previous analyses assuming that undefined amino acids translate stops showed greater divergence between nuclear and mitochondrial genetic codes. Here, three independent methods converge on which amino acids translated stops at split between nuclear and mitochondrial genetic codes: (a) alignment-free genetic code comparisons inserting different amino acids at stops; (b) alignment-based blast analyses of hypothetical peptides translated from non-coding mitochondrial sequences, inserting different amino acids at stops; (c) biases in amino acid insertions at stops in proteomic data. Hence short-term protein evolution models reconstruct long-term genetic code evolution. Mitochondria reassign stops to amino acids otherwise inserted at stops by codon-anticodon mismatches (near-cognate tRNAs). Hence dual function (translation termination and translation by codon-anticodon mismatch) precedes mitochondrial reassignments of stops to amino acids. Stop ambiguity increases coded information, compensates endocellular mitogenome reduction. Mitochondrial codon reassignments might prevent viral infections. Copyright © 2018 Elsevier B.V. All rights reserved.

High-resolution melting analysis of gyrA codon 84 and grlA codon 80 mutations conferring resistance to fluoroquinolones in Staphylococcus pseudintermedius isolates from canine clinical samples.

PubMed

Loiacono, Monica; Martino, Piera A; Albonico, Francesca; Dell'Orco, Francesca; Ferretti, Manuela; Zanzani, Sergio; Mortarino, Michele

2017-09-01

Staphylococcus pseudintermedius is an opportunistic pathogen of dogs and cats. A high-resolution melting analysis (HRMA) protocol was designed and tested on 42 clinical isolates with known fluoroquinolone (FQ) susceptibility and gyrA codon 84 and grlA codon 80 mutation status. The HRMA approach was able to discriminate between FQ-sensitive and FQ-resistant strains and confirmed previous reports that the main mutation site associated with FQ resistance in S. pseudintermedius is located at position 251 (Ser84Leu) of gyrA. Routine, HRMA-based FQ susceptibility profiles may be a valuable tool to guide therapy. The FQ resistance-predictive power of the assay should be tested in a significantly larger number of isolates.

Complete mitochondrial genome of Chocolate Pansy, Junonia iphita (Lepidoptera: Nymphalidae: Nymphalinae).

PubMed

Vanlalruati, Catherine; Mandal, Surajit De; Gurusubramanian, Guruswami; Senthil Kumar, Nachimuthu

2016-07-01

The complete mitochondrial genome of Junonia iphita was determined to be 15,433 bp in length, including 37 typical mitochondrial genes and an AT-rich region. All the protein coding genes (PCGs) are initiated by typical ATN codons, except cox1 gene that is by CGA codon. Eight genes use complete termination codon (TAA), whereas the cox1, cox2 and nad5 genes end with single T; nad4 and nad1 ends with stop codon TA. All the tRNA show secondary cloverleaf structures except trnS1 (AGN). The A + T rich region is 546 bp in length containing ATAGA motif followed by a 18 bp poly-T stretch, two microsatellite-like (TA)9 elements and 8 bp poly-A stretch immediately upstream of trnM gene.

uORFs with unusual translational start codons autoregulate expression of eukaryotic ornithine decarboxylase homologs

PubMed Central

Ivanov, Ivaylo P.; Loughran, Gary; Atkins, John F.

2008-01-01

In a minority of eukaryotic mRNAs, a small functional upstream ORF (uORF), often performing a regulatory role, precedes the translation start site for the main product(s). Here, conserved uORFs in numerous ornithine decarboxylase homologs are identified from yeast to mammals. Most have noncanonical evolutionarily conserved start codons, the main one being AUU, which has not been known as an initiator for eukaryotic chromosomal genes. The AUG-less uORF present in mouse antizyme inhibitor, one of the ornithine decarboxylase homologs in mammals, mediates polyamine-induced repression of the downstream main ORF. This repression is part of an autoregulatory circuit, and one of its sensors is the AUU codon, which suggests that translation initiation codon identity is likely used for regulation in eukaryotes. PMID:18626014

Effects of Cabin Upsets on Adsorption Columns for Air Revitalization

NASA Technical Reports Server (NTRS)

LeVan, M. Douglas

1999-01-01

The National Aeronautics and Space Administration (NASA) utilizes adsorption technology as part of contaminant removal systems designed for long term missions. A variety of trace contaminants can be effectively removed from gas streams by adsorption onto activated carbon. An activated carbon adsorption column meets NASA's requirements of a lightweight and efficient means of controlling trace contaminant levels aboard spacecraft and space stations. The activated carbon bed is part of the Trace Contaminant Control System (TCCS) which is utilized to purify the cabin atmosphere. TCCS designs oversize the adsorption columns to account for irregular fluctuations in cabin atmospheric conditions. Variations in the cabin atmosphere include changes in contaminant concentrations, temperature, and relative humidity. Excessively large deviations from typical conditions can result from unusual crew activity, equipment malfunctions, or even fires. The research carried out under this award focussed in detail on the effects of cabin upsets on the performance of activated carbon adsorption columns. Both experiments and modeling were performed with an emphasis on the roll of a change in relative humidity on adsorption of trace contaminants. A flow through fixed-bed apparatus was constructed at the NASA Ames Research Center, and experiments were performed there by W. Scot Appel under the direction of Dr. John E. Finn. Modeling work was performed at the University of Virginia and at Vanderbilt University by W. Scot Appel under the direction of M. Douglas LeVan. All three participants collaborated in all of the various phases of the research. The most comprehensive document describing the research is the Ph.D. dissertation of W. Scot Appel. Results have been published in several papers and presented in talks at technical conferences. All documents have been transmitted to Dr. John E. Finn.

Wounding response in xylem of Scots pine seedlings shows wide genetic variation and connection with the constitutive defence of heartwood.

PubMed

Harju, Anni M; Venäläinen, Martti; Laakso, Tapio; Saranpää, Pekka

2009-01-01

In this greenhouse experiment, 3-year-old Scots pine (Pinus sylvestris L.) seedlings were wounded by drilling holes through the stem. In the xylem next to the wound, the concentration of resin acids (RAC) increased, and the production of extractives typical for heartwood (stilbenes) and knotwood (stilbenes and lignans) of mature trees was induced. The induced stilbenes were pinosylvin (PS) and pinosylvin monomethyl ether (PSM), and the lignans nortrachelogenin (NTG) and matairesinol (MR). There was positive phenotypic correlation between concentrations of the different extractives. Except for the RAC, the extractive concentrations showed no correlation with the size of the seedlings. The treated seedlings belonged to half-sib families, which enabled the estimation of the genetic parameters for the response variables. The proportion of heritable variation (heritability, h(2)) in the concentration of PS, NTG and MR varied between 0.71 and 1.03, whereas for PSM and RAC the heritability was lower (0.35 and 0.31). Genetic correlation was significant between PS and PSM (r = 0.55, P = 0.018), and between NTG and MR (r = 0.50, P = 0.033). Heritabilities were also estimated on the basis of the regression of the offspring on their mothers h(2)(0P). These estimates were assessed for the concentration of PS, PSM and RAC in the wound response area of the seedlings and correspondingly in the heartwood of their mothers. The heritability was highest for the concentration of PS h(2)(0P). The findings of this study support the suggestion that the wounding of Scots pine seedlings may facilitate the development of an early testing method for breeding heartwood durability.

Tree stem diameter variations and transpiration in Scots pine: an analysis using a dynamic sap flow model.

PubMed

Perämäki, M; Nikinmaa, E; Sevanto, S; Ilvesniemi, H; Siivola, E; Hari, P; Vesala, T

2001-08-01

A dynamic model for simulating water flow in a Scots pine (Pinus sylvestris L.) tree was developed. The model is based on the cohesion theory and the assumption that fluctuating water tension driven by transpiration, together with the elasticity of wood tissue, causes variations in the diameter of a tree stem and branches. The change in xylem diameter can be linked to water tension in accordance with Hookeâ s law. The model was tested against field measurements of the diurnal xylem diameter change at different heights in a 37-year-old Scots pine at Hyytiälä, southern Finland (61 degrees 51' N, 24 degrees 17' E, 181 m a.s.l.). Shoot transpiration and soil water potential were input data for the model. The biomechanical and hydraulic properties of wood and fine root hydraulic conductance were estimated from simulated and measured stem diameter changes during the course of 1 day. The estimated parameters attained values similar to literature values. The ratios of estimated parameters to literature values ranged from 0.5 to 0.9. The model predictions (stem diameters at several heights) were in close agreement with the measurements for a period of 6 days. The time lag between changes in transpiration rate and in sap flow rate at the base of the tree was about half an hour. The analysis showed that 40% of the resistance between the soil and the top of the tree was located in the rhizosphere. Modeling the water tension gradient and consequent woody diameter changes offer a convenient means of studying the link between wood hydraulic conductivity and control of transpiration.

Limited Growth Recovery after Drought-Induced Forest Dieback in Very Defoliated Trees of Two Pine Species

PubMed Central

Guada, Guillermo; Camarero, J. Julio; Sánchez-Salguero, Raúl; Cerrillo, Rafael M. Navarro

2016-01-01

Mediterranean pine forests display high resilience after extreme climatic events such as severe droughts. However, recent dry spells causing growth decline and triggering forest dieback challenge the capacity of some forests to recover following major disturbances. To describe how resilient the responses of forests to drought can be, we quantified growth dynamics in plantations of two pine species (Scots pine, black pine) located in south-eastern Spain and showing drought-triggered dieback. Radial growth was characterized at inter- (tree-ring width) and intra-annual (xylogenesis) scales in three defoliation levels. It was assumed that the higher defoliation the more negative the impact of drought on tree growth. Tree-ring width chronologies were built and xylogenesis was characterized 3 years after the last severe drought occurred. Annual growth data and the number of tracheids produced in different stages of xylem formation were related to climate data at several time scales. Drought negatively impacted growth of the most defoliated trees in both pine species. In Scots pine, xylem formation started earlier in the non-defoliated than in the most defoliated trees. Defoliated trees presented the shortest duration of the radial-enlargement phase in both species. On average the most defoliated trees formed 60% of the number of mature tracheids formed by the non-defoliated trees in both species. Since radial enlargement is the xylogenesis phase most tightly related to final growth, this explains why the most defoliated trees grew the least due to their altered xylogenesis phases. Our findings indicate a very limited resilience capacity of drought-defoliated Scots and black pines. Moreover, droughts produce legacy effects on xylogenesis of highly defoliated trees which could not recover previous growth rates and are thus more prone to die. PMID:27066053

Defoliating Insect Mass Outbreak Affects Soil N Fluxes and Tree N Nutrition in Scots Pine Forests

PubMed Central

Grüning, Maren M.; Simon, Judy; Rennenberg, Heinz; l-M-Arnold, Anne

2017-01-01

Biotic stress by mass outbreaks of defoliating pest insects does not only affect tree performance by reducing its photosynthetic capacity, but also changes N cycling in the soil of forest ecosystems. However, how insect induced defoliation affects soil N fluxes and, in turn, tree N nutrition is not well-studied. In the present study, we quantified N input and output fluxes via dry matter input, throughfall, and soil leachates. Furthermore, we investigated the effects of mass insect herbivory on tree N acquisition (i.e., organic and inorganic 15N net uptake capacity of fine roots) as well as N pools in fine roots and needles in a Scots pine (Pinus sylvestris L.) forest over an entire vegetation period. Plots were either infested by the nun moth (Lymantria monacha L.) or served as controls. Our results show an increased N input by insect feces, litter, and throughfall at the infested plots compared to controls, as well as increased leaching of nitrate. However, the additional N input into the soil did not increase, but reduce inorganic and organic net N uptake capacity of Scots pine roots. N pools in the fine roots and needles of infested trees showed an accumulation of total N, amino acid-N, protein-N, and structural N in the roots and the remaining needles as a compensatory response triggered by defoliation. Thus, although soil N availability was increased via surplus N input, trees did not respond with an increased N acquisition, but rather invested resources into defense by accumulation of amino acid-N and protein-N as a survival strategy. PMID:28638396

Secondary metabolite concentrations and terpene emissions of Scots pine xylem after long-term forest fertilization.

PubMed

Turtola, S; Manninen, A M; Holopainen, J K; Levula, T; Raitio, H; Kainulainen, P

2002-01-01

Secondary compounds are known to be associated with the resistance of conifer xylem against insects and fungi. The effects of long-term forest fertilization with nitrogen (N) or with N, calcium (Ca), and phosphorus (P) on secondary compounds in the xylem of 50-yr-old Scots pine (Pinus sylvestris L.) trees were examined. Xylem samples were collected from trees growing in three locations in southern Finland: Vilppula, Padasjoki, and Punkaharju. Forests were fertilized every fifth (Vilppula and Padasjoki) or tenth (Punkaharju) year since the 1950s. We compared concentrations of individual and total monoterpenes and resin acids in the heartwood and sapwood of Scots pine. Terpene emissions were analyzed from the sapwood and total phenolics from the heartwood. Fertilization did not have any significant effect on the concentrations and emissions of xylem monoterpenes. Concentrations of several individual terpenes in sapwood were positively correlated with the corresponding terpene emission. The concentrations of individual resin acids (i.e., abietic and dehydroabietic) decreased significantly in Punkaharju, but increased in the sapwood of N-fertilized trees compared with control ones at Padasjoki and Vilppula. The concentrations of resin acids in the heartwood were not significantly affected by fertilization. Both fertilization treatments decreased the total phenolic concentrations in the heartwood of trees growing in Padasjoki. There was a significant positive correlation between the total phenolics and total resin acid concentration. Overall, resin acids and phenolics seemed be more responsive than monoterpenes to N treatment. These results suggest that forest fertilization might cause slight changes in secondary compound concentrations of xylem, and thus might have significance in the decay resistance of wood.

A dual stable-isotope approach to analyse the linkages between tree water fluxes and soil water pools in a Mediterranean mountain catchment

NASA Astrophysics Data System (ADS)

Llorens, Pilar; Cayuela, Carles; Sánchez-Costa, Elisenda; Gallart, Francesc; Latron, Jérôme

2017-04-01

This work uses a dual isotope-based approach (18O, 2H) to examine the mixing of water in the soil and the linkages between tree water fluxes and soil water pools in a Mediterranean mountain catchment (Vallcebre Research Catchments, NE Spain, 42° 12'N, 1° 49'E). Since May 2015, water-isotopes have been monitored in rainfall, throughfall and stemflow below a Scots pine stand and in stream water at the Can Vila (0.56 km2) catchment outlet. Moreover, fortnightly (From May to December 2015) soil samples (10, 20, 30, 50 and 100 cm), xylem samples (3 Scots pines) and mobile soil water samples in low-suction lysimeters (20, 50 and 100 cm) and in a piezometer (150-300 cm deep) were collected at the same stand. Water from soil and xylem samples was extracted by cryogenic vacuum distillation and isotope analyses were obtained by infrared spectroscopy. All this information has been combined with continuous measurement of meteorological, soil moisture and water potential, piezometric levels and hydrological variables at the stand and catchment scales. Stable isotopes ratios of bound soil water fell below the local meteoric water line (LMWL), with more evaporative enrichment in the shallow horizons. On the contrary, mobile soil water (low suction lysimeters) and groundwater fell along the LMWL, well mixed with stream water. The differences observed between these two water pools remained similar during the whole study period. Stable isotopes ratios indicate that Scots pine trees use shallow bound soil water during the whole study period. No marked changes in depth of water uptake were observed, presumably due to the availability of water in the shallow horizons, even during the summer months.

Plasticity in hydraulic architecture of Scots pine across Eurasia.

PubMed

Poyatos, R; Martínez-Vilalta, J; Cermák, J; Ceulemans, R; Granier, A; Irvine, J; Köstner, B; Lagergren, F; Meiresonne, L; Nadezhdina, N; Zimmermann, R; Llorens, P; Mencuccini, M

2007-08-01

Widespread tree species must show physiological and structural plasticity to deal with contrasting water balance conditions. To investigate these plasticity mechanisms, a meta-analysis of Pinus sylvestris L. sap flow and its response to environmental variables was conducted using datasets from across its whole geographical range. For each site, a Jarvis-type, multiplicative model was used to fit the relationship between sap flow and photosynthetically active radiation, vapour pressure deficit (D) and soil moisture deficit (SMD); and a logarithmic function was used to characterize the response of stomatal conductance (G(s)) to D. The fitted parameters of those models were regressed against climatic variables to study the acclimation of Scots pine to dry/warm conditions. The absolute value of sap flow and its sensitivity to D and SMD increased with the average summer evaporative demand. However, relative sensitivity of G(s) to D (m/G (s,ref), where m is the slope and G(s,ref) is reference G(s) at D = 1 kPa) did not increase with evaporative demand across populations, and transpiration per unit leaf area at a given D increased accordingly in drier/warmer climates. This physiological plasticity was linked to the previously reported climate- and size-related structural acclimation of leaf to sapwood area ratios. G (s,ref), and its absolute sensitivity to D(m), tended to decrease with age/height of the trees as previously reported for other pine species. It is unclear why Scots pines have higher transpiration rates at drier/warmer sites, at the expense of lower water-use efficiency. In any case, our results suggest that these structural adjustments may not be enough to prevent lower xylem tensions at the driest sites.

Nine Years of Irrigation Cause Vegetation and Fine Root Shifts in a Water-Limited Pine Forest

PubMed Central

Herzog, Claude; Steffen, Jan; Graf Pannatier, Elisabeth; Hajdas, Irka; Brunner, Ivano

2014-01-01

Scots pines (Pinus sylvestris L.) in the inner-Alpine dry valleys of Switzerland have suffered from increased mortality during the past decades, which has been caused by longer and more frequent dry periods. In addition, a proceeding replacement of Scots pines by pubescent oaks (Quercus pubescens Willd.) has been observed. In 2003, an irrigation experiment was performed to track changes by reducing drought pressure on the natural pine forest. After nine years of irrigation, we observed major adaptations in the vegetation and shifts in Scots pine fine root abundance and structure. Irrigation permitted new plant species to assemble and promote canopy closure with a subsequent loss of herb and moss coverage. Fine root dry weight increased under irrigation and fine roots had a tendency to elongate. Structural composition of fine roots remained unaffected by irrigation, expressing preserved proportions of cellulose, lignin and phenolic substances. A shift to a more negative δ13C signal in the fine root C indicates an increased photosynthetic activity in irrigated pine trees. Using radiocarbon (14C) measurement, a reduced mean age of the fine roots in irrigated plots was revealed. The reason for this is either an increase in newly produced fine roots, supported by the increase in fine root biomass, or a reduced lifespan of fine roots which corresponds to an enhanced turnover rate. Overall, the responses belowground to irrigation are less conspicuous than the more rapid adaptations aboveground. Lagged and conservative adaptations of tree roots with decadal lifespans are challenging to detect, hence demanding for long-term surveys. Investigations concerning fine root turnover rate and degradation processes under a changing climate are crucial for a complete understanding of C cycling. PMID:24802642

Testing phenotypic trade-offs in the chemical defence strategy of Scots pine under growth-limiting field conditions.

PubMed

Villari, Caterina; Faccoli, Massimo; Battisti, Andrea; Bonello, Pierluigi; Marini, Lorenzo

2014-09-01

Plants protect themselves from pathogens and herbivores through fine-tuned resource allocation, including trade-offs among resource investments to support constitutive and inducible defences. However, empirical research, especially concerning conifers growing under natural conditions, is still scarce. We investigated the complexity of constitutive and induced defences in a natural Scots pine (Pinus sylvestris L.) stand under growth-limiting conditions typical of alpine environments. Phenotypic trade-offs at three hierarchical levels were tested by investigating the behaviour of phenolic compounds and terpenoids of outer bark and phloem. We tested resource-derived phenotypic correlations between (i) constitutive and inducible defences vs tree ring growth, (ii) different constitutive defence metabolites and (iii) constitutive concentration and inducible variation of individual metabolites. Tree ring growth was positively correlated only with constitutive concentration of total terpenoids, and no overall phenotypic trade-offs between different constitutive defensive metabolites were found. At the lowest hierarchical level tested, i.e., at the level of relationship between constitutive and inducible variation of individual metabolites, we found that different compounds displayed different behaviours; we identified five different defensive metabolite response types, based on direction and strength of the response, regardless of tree age and growth rate. Therefore, under growth-limiting field conditions, Scots pine appears to utilize varied and complex outer bark and phloem defence chemistry, in which only part of the constitutive specialized metabolism is influenced by tree growth, and individual components do not appear to be expressed in a mutually exclusive manner in either constitutive or inducible metabolism. © The Author 2014. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

Polymorphism at codon 36 of the p53 gene.

PubMed

Felix, C A; Brown, D L; Mitsudomi, T; Ikagaki, N; Wong, A; Wasserman, R; Womer, R B; Biegel, J A

1994-01-01

A polymorphism at codon 36 in exon 4 of the p53 gene was identified by single strand conformation polymorphism (SSCP) analysis and direct sequencing of genomic DNA PCR products. The polymorphic allele, present in the heterozygous state in genomic DNAs of four of 100 individuals (4%), changes the codon 36 CCG to CCA, eliminates a FinI restriction site and creates a BccI site. Including this polymorphism there are four known polymorphisms in the p53 coding sequence.

Genomic adaptation of the ISA virus to Salmo salar codon usage

PubMed Central

2013-01-01

Background The ISA virus (ISAV) is an Orthomyxovirus whose genome encodes for at least 10 proteins. Low protein identity and lack of genetic tools have hampered the study of the molecular mechanism behind its virulence. It has been shown that viral codon usage controls several processes such as translational efficiency, folding, tuning of protein expression, antigenicity and virulence. Despite this, the possible role that adaptation to host codon usage plays in virulence and viral evolution has not been studied in ISAV. Methods Intergenomic adaptation between viral and host genomes was calculated using the codon adaptation index score with EMBOSS software and the Kazusa database. Classification of host genes according to GeneOnthology was performed using Blast2go. A non parametric test was applied to determine the presence of significant correlations among CAI, mortality and time. Results Using the codon adaptation index (CAI) score, we found that the encoding genes for nucleoprotein, matrix protein M1 and antagonist of Interferon I signaling (NS1) are the ISAV genes that are more adapted to host codon usage, in agreement with their requirement for production of viral particles and inactivation of antiviral responses. Comparison to host genes showed that ISAV shares CAI values with less than 0.45% of Salmo salar genes. GeneOntology classification of host genes showed that ISAV genes share CAI values with genes from less than 3% of the host biological process, far from the 14% shown by Influenza A viruses and closer to the 5% shown by Influenza B and C. As well, we identified a positive correlation (p<0.05) between CAI values of a virus and the duration of the outbreak disease in given salmon farms, as well as a weak relationship between codon adaptation values of PB1 and the mortality rates of a set of ISA viruses. Conclusions Our analysis shows that ISAV is the least adapted viral Salmo salar pathogen and Orthomyxovirus family member less adapted to host codon usage, avoiding the general behavior of host genes. This is probably due to its recent emergence among farmed Salmon populations. PMID:23829271

Genomic adaptation of the ISA virus to Salmo salar codon usage.

PubMed

Tello, Mario; Vergara, Francisco; Spencer, Eugenio

2013-07-05

The ISA virus (ISAV) is an Orthomyxovirus whose genome encodes for at least 10 proteins. Low protein identity and lack of genetic tools have hampered the study of the molecular mechanism behind its virulence. It has been shown that viral codon usage controls several processes such as translational efficiency, folding, tuning of protein expression, antigenicity and virulence. Despite this, the possible role that adaptation to host codon usage plays in virulence and viral evolution has not been studied in ISAV. Intergenomic adaptation between viral and host genomes was calculated using the codon adaptation index score with EMBOSS software and the Kazusa database. Classification of host genes according to GeneOnthology was performed using Blast2go. A non parametric test was applied to determine the presence of significant correlations among CAI, mortality and time. Using the codon adaptation index (CAI) score, we found that the encoding genes for nucleoprotein, matrix protein M1 and antagonist of Interferon I signaling (NS1) are the ISAV genes that are more adapted to host codon usage, in agreement with their requirement for production of viral particles and inactivation of antiviral responses. Comparison to host genes showed that ISAV shares CAI values with less than 0.45% of Salmo salar genes. GeneOntology classification of host genes showed that ISAV genes share CAI values with genes from less than 3% of the host biological process, far from the 14% shown by Influenza A viruses and closer to the 5% shown by Influenza B and C. As well, we identified a positive correlation (p<0.05) between CAI values of a virus and the duration of the outbreak disease in given salmon farms, as well as a weak relationship between codon adaptation values of PB1 and the mortality rates of a set of ISA viruses. Our analysis shows that ISAV is the least adapted viral Salmo salar pathogen and Orthomyxovirus family member less adapted to host codon usage, avoiding the general behavior of host genes. This is probably due to its recent emergence among farmed Salmon populations.

High level production of β-galactosidase exhibiting excellent milk-lactose degradation ability from Aspergillus oryzae by codon and fermentation optimization.

PubMed

Zhao, Qianqian; Liu, Fei; Hou, Zhongwen; Yuan, Chao; Zhu, Xiqiang

2014-03-01

A β-galactosidase gene from Aspergillus oryzae was engineered utilizing codon usage optimization to be constitutively and highly expressed in the Pichia pastoris SMD1168H strain in a high-cell-density fermentation. After fermentation for 96 h in a 50-L fermentor using glucose and glycerol as combined carbon sources, the recombinant enzyme in the culture supernatant had an activity of 4,239.07 U mL(-1) with o-nitrophenyl-β-D-galactopyranoside as the substrate, and produced a total of extracellular protein content of 7.267 g L(-1) in which the target protein (6.24 g L(-1)) occupied approximately 86 %. The recombinant β-galactosidase exhibited an excellent lactose hydrolysis ability. With 1,000 U of the enzyme in 100 mL milk, 92.44 % lactose was degraded within 24 h at 60 °C, and the enzyme could also accomplish the hydrolysis at low temperatures of 37, 25, and 10 °C. Thus, this engineered strain had significantly higher fermentation level of A. oryzae lactase than that before optimization and the β-galactosidase may have a good application potential in whey and milk industries.

Culture adaptation of malaria parasites selects for convergent loss-of-function mutants.

PubMed

Claessens, Antoine; Affara, Muna; Assefa, Samuel A; Kwiatkowski, Dominic P; Conway, David J

2017-01-24

Cultured human pathogens may differ significantly from source populations. To investigate the genetic basis of laboratory adaptation in malaria parasites, clinical Plasmodium falciparum isolates were sampled from patients and cultured in vitro for up to three months. Genome sequence analysis was performed on multiple culture time point samples from six monoclonal isolates, and single nucleotide polymorphism (SNP) variants emerging over time were detected. Out of a total of five positively selected SNPs, four represented nonsense mutations resulting in stop codons, three of these in a single ApiAP2 transcription factor gene, and one in SRPK1. To survey further for nonsense mutants associated with culture, genome sequences of eleven long-term laboratory-adapted parasite strains were examined, revealing four independently acquired nonsense mutations in two other ApiAP2 genes, and five in Epac. No mutants of these genes exist in a large database of parasite sequences from uncultured clinical samples. This implicates putative master regulator genes in which multiple independent stop codon mutations have convergently led to culture adaptation, affecting most laboratory lines of P. falciparum. Understanding the adaptive processes should guide development of experimental models, which could include targeted gene disruption to adapt fastidious malaria parasite species to culture.

β-Glucuronidase as a Sensitive and Versatile Reporter in Actinomycetes ▿

PubMed Central

Myronovskyi, Maksym; Welle, Elisabeth; Fedorenko, Viktor; Luzhetskyy, Andriy

2011-01-01

Here we describe a versatile and sensitive reporter system for actinomycetes that is based on gusA, which encodes the β-glucuronidase enzyme. A series of gusA-containing transcriptional and translational fusion vectors were constructed and utilized to study the regulatory cascade of the phenalinolactone biosynthetic gene cluster. Furthermore, these vectors were used to study the efficiency of translation initiation at the ATG, GTG, TTG, and CTG start codons. Surprisingly, constructs using a TTG start codon showed the best activity, whereas those using ATG or GTG were approximately one-half or one-third as active, respectively. The CTG fusion showed only 5% of the activity of the TTG fusion. A suicide vector, pKGLP2, carrying gusA in its backbone was used to visually detect merodiploid formation and resolution, making gene targeting in actinomycetes much faster and easier. Three regulatory genes, plaR1, plaR2, and plaR3, involved in phenalinolactone biosynthesis were efficiently replaced with an apramycin resistance marker using this system. Finally, we expanded the genetic code of actinomycetes by introducing the nonproteinogenic amino acid N-epsilon-cyclopentyloxycarbonyl-l-lysine with the GusA protein as a reporter. PMID:21685164

Application of a multi-channel microfluidic chip on the simultaneous detection of DNAs by using microbead-quantum dots.

PubMed

Le, Ngoc Tam; Kim, Jong Sung

2014-12-01

Several researches have shown that cancer is caused by genetic mutations especially in genes involved in cell growth and regulation. Ras family members are frequently found in their mutated, oncogenic forms in human tumors. Mutant RAS proteins are constitutively active, owing to reduce intrinsic GTPase activity and insensitivity to GTPase-activating protein (GAPs). In total, activating mutations in the RAS genes occur in approximately 20% of all human cancers, mainly in codon 12, 13 or 61. Activating mutations in the NRAS gene not only result in the reduction of intrinsic GTPase activity but also in the induction of resistance against molecules inducing such activity. In this paper, we reported a rapid, simple and portable method for detecting the mutant types of NRAS genes codon 12 and 61 simultaneously by using bead-quantum dots (QDs) based multi-channel microfluidic chip. Probe DNAs are conjugated to bead-QDs and packed in the pillars of channels in the microfluidic chip. After injection of target DNAs and intercalating dyes, the fluorescence quenching of QDs by intercalating dye was observed due to FRET phenomena. The platform can be effortlessly applied in other biological and clinical areas.

A mechanism for exon skipping caused by nonsense or missense mutations in BRCA1 and other genes.

PubMed

Liu, H X; Cartegni, L; Zhang, M Q; Krainer, A R

2001-01-01

Point mutations can generate defective and sometimes harmful proteins. The nonsense-mediated mRNA decay (NMD) pathway minimizes the potential damage caused by nonsense mutations. In-frame nonsense codons located at a minimum distance upstream of the last exon-exon junction are recognized as premature termination codons (PTCs), targeting the mRNA for degradation. Some nonsense mutations cause skipping of one or more exons, presumably during pre-mRNA splicing in the nucleus; this phenomenon is termed nonsense-mediated altered splicing (NAS), and its underlying mechanism is unclear. By analyzing NAS in BRCA1, we show here that inappropriate exon skipping can be reproduced in vitro, and results from disruption of a splicing enhancer in the coding sequence. Enhancers can be disrupted by single nonsense, missense and translationally silent point mutations, without recognition of an open reading frame as such. These results argue against a nuclear reading-frame scanning mechanism for NAS. Coding-region single-nucleotide polymorphisms (cSNPs) within exonic splicing enhancers or silencers may affect the patterns or efficiency of mRNA splicing, which may in turn cause phenotypic variability and variable penetrance of mutations elsewhere in a gene.

Arctic Technology Evaluation 2014 Oil-in-Ice Demonstration Report

DTIC Science & Technology

2015-03-01

Spills in Ice”, 2014 International Oil Spill Conference, Savannah, GA, May, 2014. Hansen, Kurt A., Scot T. Trip, Rich L. Hansen (2014) “Evaluating...Operations”, Woods Hole Oceanographic Institution (WHOI) report to BSEE, 2013. Arctic Technology Evaluation 2014 Oil-in-Ice Demonstration Report 40

Logging residue removal after thinning in boreal forests: long-term impact on the nutrient status of Norway spruce and Scots pine needles.

PubMed

Luiro, Jukka; Kukkola, Mikko; Saarsalmi, Anna; Tamminen, Pekka; Helmisaari, Heljä-Sisko

2010-01-01

The aim of this study was to compare how conventional stem harvesting (CH) and whole-tree harvesting (WTH) in the first, and in some cases also in the second, thinning affect the needle nutrient status of Scots pine (Pinus sylvestris L.) and Norway spruce (Picea abies (L.) Karst.) stands in Finland. A series of 12 long-term field experiments was studied. The experiments were established during 1978-86. The effects of logging residue removal after thinnings on the needle nutrient concentrations were generally minor and without any overall trends, but there were differences between experiments. Trees tend to maintain their current needle nutrient concentrations at the same level by re-utilizing the nutrients stored in the older tissues and by changing C allocation in the whole tree. Thus, needle analysis should be combined with stem growth data in order to achieve a more comprehensive understanding of the effects of WTH on the nutrient status of trees.

Injection molding lens metrology using software configurable optical test system

NASA Astrophysics Data System (ADS)

Zhan, Cheng; Cheng, Dewen; Wang, Shanshan; Wang, Yongtian

2016-10-01

Optical plastic lens produced by injection molding machine possesses numerous advantages of light quality, impact resistance, low cost, etc. The measuring methods in the optical shop are mainly interferometry, profile meter. However, these instruments are not only expensive, but also difficult to alignment. The software configurable optical test system (SCOTS) is based on the geometry of the fringe refection and phase measuring deflectometry method (PMD), which can be used to measure large diameter mirror, aspheric and freeform surface rapidly, robustly, and accurately. In addition to the conventional phase shifting method, we propose another data collection method called as dots matrix projection. We also use the Zernike polynomials to correct the camera distortion. This polynomials fitting mapping distortion method has not only simple operation, but also high conversion precision. We simulate this test system to measure the concave surface using CODE V and MATLAB. The simulation results show that the dots matrix projection method has high accuracy and SCOTS has important significance for on-line detection in optical shop.

Characterization of Scots pine stump-root biomass as feed-stock for gasification.

PubMed

Eriksson, Daniel; Weiland, Fredrik; Hedman, Henry; Stenberg, Martin; Öhrman, Olov; Lestander, Torbjörn A; Bergsten, Urban; Öhman, Marcus

2012-01-01

The main objective was to explore the potential for gasifying Scots pine stump-root biomass (SRB). Washed thin roots, coarse roots, stump heartwood and stump sapwood were characterized (solid wood, milling and powder characteristics) before and during industrial processing. Non-slagging gasification of the SRB fuels and a reference stem wood was successful, and the gasification parameters (synthesis gas and bottom ash characteristics) were similar. However, the heartwood fuel had high levels of extractives (≈19%) compared to the other fuels (2-8%) and thereby ≈16% higher energy contents but caused disturbances during milling, storage, feeding and gasification. SRB fuels could be sorted automatically according to their extractives and moisture contents using near-infrared spectroscopy, and their amounts and quality in forests can be predicted using routinely collected stand data, biomass functions and drill core analyses. Thus, SRB gasification has great potential and the proposed characterizations exploit it. Copyright © 2011 Elsevier Ltd. All rights reserved.

Effect of long-term forest fertilization on Scots pine xylem quality and wood borer performance.

PubMed

Heijari, Juha; Nerg, Anne-Marja; Kainulainen, Pirjo; Noldt, Uwe; Levula, Teuvo; Raitio, Hannu; Holopainen, Jarmo K

2008-01-01

We tested whether changes in long-term nutrient availability would affect the xylem quality and characteristics of Scots pine trees as a food source for the larvae of the xylophagous wood borer Hylotrupes bajulus L. (Cerambycidae). We looked for an effect of host plant growth and xylem structural traits on H. bajulus larval performance, and looked for delayed effects of long-term forest fertilization on xylem chemical quality. In general, larval performance was dependent on larval developmental stage. However, the growth of larvae also varied with host plant quality (increases in the concentration of nitrogen and carbon-based secondary compounds of xylem were correlated with a decrease in the larval growth rate). The greater annual growth of trees reduced tracheid length and correlated positively with second-instar H. bajulus growth rate. This is consistent with the hypothesis that intrinsic growth patterns of host plants influence the development of the xylophagous wood borer H. bajulus.

Needle and stem wood production in Scots pine (Pinus sylvestris) trees of different age, size and competitive status.

PubMed

Vanninen, Petteri; Mäkelä, Annikki

2000-04-01

We studied effects of tree age, size and competitive status on foliage and stem production of 43 Scots pine (Pinus sylvestris L.) trees in southern Finland. The tree attributes related to competition included foliage density, crown ratio and height/diameter ratio. Needle mass was considered to be the primary cause of growth through photosynthesis. Both stem growth and foliage growth were strongly correlated with foliage mass. Consequently, differences in growth allocation between needles and stem wood in trees of different age, size, or position were small. However, increasing relative height increased the sum of stem growth and foliage growth per unit foliage mass, indicating an effect of available light. Suppressed trees seemed to allocate more growth to stem wood than dominant trees, and their stem growth per unit foliage mass was larger. Similarly, trees in dense stands allocated more growth to stem wood than trees in sparse stands. The results conformed to the pipe model theory but seemed to contradict the priority principle of allocation.

Contribution of ambient ozone to Scots pine defoliation and reduced growth in the Central European forests: a Lithuanian case study.

PubMed

Augustaitis, Algirdas; Bytnerowicz, Andrzej

2008-10-01

The study aimed to explore if changes in crown defoliation and stem growth of Scots pines (Pinus sylvestris L.) could be related to changes in ambient ozone (O(3)) concentration in central Europe. To meet this objective the study was performed in 3 Lithuanian national parks, close to the ICP integrated monitoring stations from which data on meteorology and pollution were provided. Contribution of peak O(3) concentrations to the integrated impact of acidifying compounds and meteorological parameters on pine stem growth was found to be more significant than its contribution to the integrated impact of acidifying compounds and meteorological parameters on pine defoliation. Findings of the study provide statistical evidence that peak concentrations of ambient O(3) can have a negative impact on pine tree crown defoliation and stem growth reduction under field conditions in central and northeastern Europe where the AOT40 values for forests are commonly below their phytotoxic levels.

Long term changes in atmospheric N and S throughfall deposition and effects on soil solution chemistry in a Scots pine forest in the Netherlands.

PubMed

Boxman, Andries W; Peters, Roy C J H; Roelofs, Jan G M

2008-12-01

In a Scots pine forest the throughfall deposition and the chemical composition of the soil solution was monitored since 1984. (Inter)national legislation measures led to a reduction of the deposition of nitrogen and sulphur. The deposition of sulphur has decreased by approximately 65%. The total mineral-nitrogen deposition has decreased by ca. 25%, which is mainly due to a reduction in ammonium-N deposition (-40%), since nitrate-N deposition has increased (+50%). The nitrogen concentration in the upper mineral soil solution at 10 cm depth has decreased, leading to an improved nutritional balance, which may result in improved tree vitality. In the drainage water at 90 cm depth the fluxes of NO3(-) and SO4(2-) have decreased, resulting in a reduced leeching of accompanying base cations, thus preserving nutrients in the ecosystem. It may take still several years, however, before this will meet the prerequisite of a sustainable ecosystem.

The PEP-3-KLH (CDX-110) vaccine in glioblastoma multiforme patients

PubMed Central

Heimberger, Amy B.; Sampson, John H

2009-01-01

Conventional therapies for glioblastoma multiforme (GBM) fail to target tumor cells exclusively resulting in non-specific toxicity. Immune targeting of tumor-specific mutations may allow for more precise eradication of neoplastic cells. The epidermal growth factor receptor variant III (EGFRvIII) is a tumor-specific mutation that is widely expressed on GBM and other neoplasms and its expression enhances tumorigenicity. This in-frame deletion mutation splits a codon resulting in a novel glycine at the fusion junction producing a tumor-specific epitope target for cellular or humoral immunotherapy. We have previously shown that vaccination with a peptide that spans the EGFRvIII fusion junction (PEPvIII-KLH/CDX-110) is an efficacious immunotherapy in syngeneic murine models. In this review, we summarize our results in GBM patients targeting this mutation in multiple, multi-institutional Phase II immunotherapy trials. These trials demonstrated that a selected population of GBM patients who received the vaccines targeting EGFRvIII had an unexpectedly long survival time. Further therapeutic strategies and potential pitfalls using this approach are discussed. PMID:19591631

Identification of hundreds of novel UPF1 target transcripts by direct determination of whole transcriptome stability

PubMed Central

Tani, Hidenori; Imamachi, Naoto; Salam, Kazi Abdus; Mizutani, Rena; Ijiri, Kenichi; Irie, Takuma; Yada, Tetsushi; Suzuki, Yutaka; Akimitsu, Nobuyoshi

2012-01-01

UPF1 eliminates aberrant mRNAs harboring premature termination codons, and regulates the steady-state levels of normal physiological mRNAs. Although genome-wide studies of UPF1 targets performed, previous studies did not distinguish indirect UPF1 targets because they could not determine UPF1-dependent altered RNA stabilities. Here, we measured the decay rates of the whole transcriptome in UPF1-depleted HeLa cells using BRIC-seq, an inhibitor-free method for directly measuring RNA stability. We determined the half-lives and expression levels of 9,229 transcripts. An amount of 785 transcripts were stabilized in UPF1-depleted cells. Among these, the expression levels of 76 transcripts were increased, but those of the other 709 transcripts were not altered. RNA immunoprecipitation showed UPF1 bound to the stabilized transcripts, suggesting that UPF1 directly degrades the 709 transcripts. Many UPF1 targets in this study were newly identified. This study clearly demonstrates that direct determination of RNA stability is a powerful approach for identifying targets of RNA degradation factors. PMID:23064114

Cytochrome oxidase subunit II gene in mitochondria of Oenothera has no intron

PubMed Central

Hiesel, Rudolf; Brennicke, Axel

1983-01-01

The cytochrome oxidase subunit II gene has been localized in the mitochondrial genome of Oenothera berteriana and the nucleotide sequence has been determined. The coding sequence contains 777 bp and, unlike the corresponding gene in Zea mays, is not interrupted by an intron. No TGA codon is found within the open reading frame. The codon CGG, as in the maize gene, is used in place of tryptophan codons of corresponding genes in other organisms. At position 742 in the Oenothera sequence the TGG of maize is changed into a CGG codon, where Trp is conserved as the amino acid in other organisms. Homologous sequences occur more than once in the mitochondrial genome as several mitochondrial DNA species hybridize with DNA probes of the cytochrome oxidase subunit II gene. ImagesFig. 5. PMID:16453484

Model for Codon Position Bias in RNA Editing

NASA Astrophysics Data System (ADS)

Liu, Tsunglin; Bundschuh, Ralf

2005-08-01

RNA editing can be crucial for the expression of genetic information via inserting, deleting, or substituting a few nucleotides at specific positions in an RNA sequence. Within coding regions in an RNA sequence, editing usually occurs with a certain bias in choosing the positions of the editing sites. In the mitochondrial genes of Physarum polycephalum, many more editing events have been observed at the third codon position than at the first and second, while in some plant mitochondria the second codon position dominates. Here we propose an evolutionary model that explains this bias as the basis of selection at the protein level. The model predicts a distribution of the three positions rather close to the experimental observation in Physarum. This suggests that the codon position bias in Physarum is mainly a consequence of selection at the protein level.

A model for codon position bias in RNA editing

NASA Astrophysics Data System (ADS)

Bundschuh, Ralf; Liu, Tsunglin

2006-03-01

RNA editing can be crucial for the expression of genetic information via inserting, deleting, or substituting a few nucleotides at specific positions in an RNA sequence. Within coding regions in an RNA sequence, editing usually occurs with a certain bias in choosing the positions of the editing sites. In the mitochondrial genes of Physarum polycephalum, many more editing events have been observed at the third codon position than at the first and second, while in some plant mitochondria the second codon position dominates. Here we propose an evolutionary model that explains this bias as the basis of selection at the protein level. The model predicts a distribution of the three positions rather close to the experimental observation in Physarum. This suggests that the codon position bias in Physarum is mainly a consequence of selection at the protein level.