Search

Ophidiomyces ophiodiicola , which belongs to the order Onygenales , is an emerging fungal pathogen of snakes in the United States. This study reports the 21.9-Mb genome sequence of an isolate of this reptilian pathogen obtained from a black racer snake in Pennsylvania.

Hybridization between distantly related organisms can facilitate rapid adaptation to novel environments, but is potentially constrained by epistatic fitness interactions among cell components. The zoonotic pathogens Campylobacter coli and C. jejuni differ from each other by around 15% at the nucleotide level, corresponding to an average of nearly 40 amino acids per protein-coding gene. Using whole genome sequencing, we show that a single C. coli lineage, which has successfully colonized an agricultural niche, has been progressively accumulating C. jejuni DNA. Members of this lineage belong to two groups, the ST-828 and ST-1150 clonal complexes. The ST-1150 complex is less frequently isolated and has undergone a substantially greater amount of introgression leading to replacement of up to 23% of the C. coli core genome as well as import of novel DNA. By contrast, the more commonly isolated ST-828 complex bacteria have 10-11% introgressed DNA, and C. jejuni and nonagricultural C. coli lineages each have

Many HIV-1 envelope-reactive antibodies shortly after HIV-1 transmission may arise from crow-reactive memory B cells previously stimulated by non-HIV-1 host or microbial antigens, The initial antibody response to HIV-1 is targeted to envelope (Env) gp41, and is nonneutralizing and ineffective in controlling viremia. To understand the origins and characteristics of gp41-binding antibodies produced shortly after HIV-1 transmission, we isolated and studied gp41-reactive plasma cells from subjects acutely infected with HIV-1. The frequencies of somatic mutations were relatively high in these gp41-reactive antibodies. Reverted unmutated ancestors of gp41-reactive antibodies derived from subjects acutely infected with HIV-1 frequently did not react with autologous HIV-1 Env; however, these antibodies were polyreactive and frequently bound to host or bacterial antigens. In one large clonal lineage of gp41-reactive antibodies, reactivity to HIV-1 Env was acquired only after somatic mutations. Polyreactive gp41-binding antibodies were also isolated from uninfected individuals. These data suggest that the majority of gp41-binding antibodies produced after acute HIV-1 infection are cross-reactive responses generated by stimulating memory B cells that have previously been activated by non–HIV-1 antigens.

While worldwide pandemic influenza A(H1N1) pdm case fatality rate (CFR) was 0.4%, Argentina's was 4.5%. A total of 34 strains from mild and severe cases were analyzed. A full genome sequencing was carried out on 26 of these, and a partial sequencing on the remaining eight. We observed no evidence that the high CFR can be attributed to direct virus changes. No evidence of re-assortment, mutations associated with resistance to antiviral drugs, or genetic drift that might contribute to virulence was observed. Although the mutation D225G associated with severity in the latest reports from the Ukraine and Norway is not observed among the Argentine strains, an amino acid change in the area (S206T) surrounding the HA receptor binding domain was observed, the same previously established worldwide.Mientras que la tasa de letalidad (CFR) para (H1N1)pdm en todo el mundo era del 0.4%, en la Argentina la mortalidad observada fue de 4.5%. La secuenciación del genoma completo de 26 cepas de virus argentinos de influenza A (H1N1)pdm de casos leves y graves y de 8 cepas secuenciadas parcialmente no mostró evidencia de que la elevada tasa de letalidad se pueda atribuir directamente a cambios en el virus. No se encontraron hallazgos de recombinación, de mutaciones asociadas con la resistencia a los medicamentos antivirales ni de variaciones genéticas que puedan contribuir a la virulencia observada. Si bien la mutación D225G asociada con la gravedad, comunicada en informes procedentes de Ucrania y Noruega, no se ha encontrado en las cepas argentinas estudiadas, se ha observado un cambio aminoacídico en la región (S206T) en torno al dominio del sitio de unión al receptor en la HA, el mismo hallado en cepas distribuidas alrededor del mundo.

Individual variation in the Ig germline gene repertoire leads to individual differences in the combinatorial diversity of the Ab repertoire, but the study of such variation has been problematic. The application of high-throughput DNA sequencing to the study of rearranged Ig genes now makes this possible. The sequencing of thousands of VDJ rearrangements from an individual, either from genomic DNA or expressed mRNA, should allow their germline IGHV, IGHD, and IGHJ repertoires to be inferred. In addition, where previously mere glimpses of diversity could be gained from sequencing studies, new large data sets should allow the rearrangement frequency of different genes and alleles to be seen with clarity. We analyzed the DNA of 108,210 human IgH chain rearrangements from 12 individuals and determined their individual IGH genotypes. The number of reportedly functional IGHV genes and allelic variants ranged from 45 to 60, principally because of variable levels of gene heterozygosity, and included 14 previously unreported IGHV polymorphisms. New polymorphisms of the IGHD3-16 and IGHJ6 genes were also seen. At heterozygous loci, remarkably different rearrangement frequencies were seen for the various IGHV alleles, and these frequencies were consistent between individuals. The specific alleles that make up an individual's Ig genotype may therefore be critical in shaping the combinatorial repertoire. The extent of genotypic variation between individuals is highlighted by an individual with aplastic anemia who appears to lack six contiguous IGHD genes on both chromosomes. These deletions significantly alter the potential expressed IGH repertoire, and possibly immune function, in this individual.

Unbiased pyrosequencing detected an astrovirus after conventional methods failed to identify the causative agent., Encephalitis is a major cause of death worldwide. Although >100 pathogens have been identified as causative agents, the pathogen is not determined for up to 75% of cases. This diagnostic failure impedes effective treatment and underscores the need for better tools and new approaches for detecting novel pathogens or determining new manifestations of known pathogens. Although astroviruses are commonly associated with gastroenteritis, they have not been associated with central nervous system disease. Using unbiased pyrosequencing, we detected an astrovirus as the causative agent for encephalitis in a 15-year-old boy with agammaglobulinemia; several laboratories had failed to identify the agent. Our findings expand the spectrum of causative agents associated with encephalitis and highlight unbiased molecular technology as a valuable tool for differential diagnosis of unexplained disease.

Characterization of arboviruses at the interface of pristine habitats and anthropogenic landscapes is crucial to comprehensive emergent disease surveillance and forecasting efforts. In context of a surveillance campaign in and around a West African rainforest, particles morphologically consistent with rhabdoviruses were identified in cell cultures infected with homogenates of trapped mosquitoes. RNA recovered from these cultures was used to derive the first complete genome sequence of a rhabdovirus isolated from Culex decens mosquitoes in Côte d'Ivoire, tentatively named Moussa virus (MOUV). MOUV shows the classical genome organization of rhabdoviruses, with five open reading frames (ORF) in a linear order. However, sequences show only limited conservation (12-33% identity at amino acid level), and ORF2 and ORF3 have no significant similarity to sequences deposited in GenBank. Phylogenetic analysis indicates a potential new species with distant relationship to Tupaia and Tibrogargan virus.

Objectives: K103N, the most common nonnucleoside reverse transcriptase inhibitor (NNRTI)–resistant mutation in patients with transmitted resistance and in patients receiving a failing NNRTIcontaining regimen, is fully susceptible to the new NNRTI, etravirine. Therefore, we sought to determine how often NNRTI-resistant mutations other than K103N occur as minority variants in plasma samples for which standard genotypic resistance testing detects K103N alone. Methods: We performed ultradeep pyrosequencing (UDPS; 454 Life Sciences a Roche Company, Branford, CT) of plasma virus samples from 13 treatment-naive and 20 NNRTI-experienced patients in whom standard genotypic resistance testing revealed K103N but no other major NNRTI-resistance mutations. Results: Samples from 0 of 13 treatment-naive patients vs. 7 of 20 patients failing an NNRTI-containing regimen had minority variants with major etravirine-associated NNRTI-resistant mutations (P = 0.03, Fisher exact test): Y181C (7.0%), Y181C (3.6%) + G190A (3.2%), L100I (14%), L100I (32%) + 190A (5.4%), K101E (3.8%) + G190A (4.9%), K101E (4.0%) + G190S (4.8%), and G190S (3.1%). Conclusions: In treatment-naive patients, UDPS did not detect additional major NNRTI-resistant mutations suggesting that etravirine may be effective in patients with transmitted K103N. In NNRTI-experienced patients, UDPS often detected additional major NNRTI-resistant mutations suggesting that etravirine may not be fully active in patients with acquired K103N.

The genome sequencing of all major food and bioenergy crops is of critical importance in the race to improve crop production to meet the future food and energy security needs of the world. Next generation sequencing technologies have brought about great improvements in sequencing throughput and cost, but do not yet allow for de novo sequencing of large repetitive genomes as found in most crop plants. We present a strategy that combines cutting edge next generation sequencing with “old school” genomics resources and allows rapid cost-effective sequencing of plant genomes.

Background. Minor (i.e.

The human distal gut harbours a vast ensemble of microbes (the microbiota) that provide important metabolic capabilities, including the ability to extract energy from otherwise indigestible dietary polysaccharides. Studies of a few unrelated, healthy adults have revealed substantial diversity in their gut communities, as measured by sequencing 16S rRNA genes, yet how this diversity relates to function and to the rest of the genes in the collective genomes of the microbiota (the gut microbiome) remains obscure. Studies of lean and obese mice suggest that the gut microbiota affects energy balance by influencing the efficiency of calorie harvest from the diet, and how this harvested energy is used and stored. Here we characterize the faecal microbial communities of adult female monozygotic and dizygotic twin pairs concordant for leanness or obesity, and their mothers, to address how host genotype, environmental exposure and host adiposity influence the gut microbiome. Analysis of 154 individuals yielded 9,920 near full-length and 1,937,461 partial bacterial 16S rRNA sequences, plus 2.14 gigabases from their microbiomes. The results reveal that the human gut microbiome is shared among family members, but that each person's gut microbial community varies in the specific bacterial lineages present, with a comparable degree of co-variation between adult monozygotic and dizygotic twin pairs. However, there was a wide array of shared microbial genes among sampled individuals, comprising an extensive, identifiable 'core microbiome' at the gene, rather than at the organismal lineage, level. Obesity is associated with phylum-level changes in the microbiota, reduced bacterial diversity and altered representation of bacterial genes and metabolic pathways. These results demonstrate that a diversity of organismal assemblages can nonetheless yield a core microbiome at a functional level, and that deviations from this core are associated with different physiological states (obese compared with lean). ©2009 Macmillan Publishers Limited. All rights reserved.

Pyrosequencing of cDNA from brains of parrots with proventricular dilatation disease (PDD), an unexplained fatal inflammatory central, autonomic, and peripheral nervous system disease, showed 2 strains of a novel Borna virus. Real-time PCR confirmed virus presence in brain, proventriculus, and adrenal gland of 3 birds with PDD but not in 4 unaffected birds.

Structural variation of the genome involves kilobase- to megabase-sized deletions, duplications, insertions, inversions, and complex combinations of rearrangements. We introduce high-throughput and massive paired-end mapping (PEM), a large-scale genome-sequencing method to identify structural variants (SVs) ∼3 kilobases (kb) or larger that combines the rescue and capture of paired ends of 3-kb fragments, massive 454 sequencing, and a computational approach to map DNA reads onto a reference genome. PEM was used to map SVs in an African and in a putatively European individual and identified shared and divergent SVs relative to the reference genome. Overall, we fine-mapped more than 1000 SVs and documented that the number of SVs among humans is much larger than initially hypothesized; many of the SVs potentially affect gene function. The breakpoint junction sequences of more than 200 SVs were determined with a novel pooling strategy and computational analysis. Our analysis provided insights into the mechanisms of SV formation in humans.

In colony collapse disorder (CCD), honey bee colonies inexplicably lose their workers. CCD has resulted in a loss of 50 to 90% of colonies in beekeeping operations across the United States. The observation that irradiated combs from affected colonies can be repopulated with naive bees suggests that infection may contribute to CCD. We used an unbiased metagenomic approach to survey microflora in CCD hives, normal hives, and imported royal jelly. Candidate pathogens were screened for significance of association with CCD by the examination of samples collected from several sites over a period of 3 years. One organism, Israeli acute paralysis virus of bees, was strongly correlated with CCD.

The proliferation of large-scale DNA-sequencing projects in recent years has driven a search for alternative methods to reduce time and cost. Here we describe a scalable, highly parallel sequencing system with raw throughput significantly greater than that of state-of-the-art capillary electrophoresis instruments. The apparatus uses a novel fibre-optic slide of individual wells and is able to sequence 25 million bases, at 99% or better accuracy, in one four-hour run. To achieve an approximately 100-fold increase in throughput over current Sanger sequencing technology, we have developed an emulsion method for DNA amplification and an instrument for sequencing by synthesis using a pyrosequencing protocol optimized for solid support and picolitre-scale volumes. Here we show the utility, throughput, accuracy and robustness of this system by shotgun sequencing and de novo assembly of the Mycoplasma genitalium genome with 96% coverage at 99.96% accuracy in one run of the machine.

The complete sequencing of the human genome and the development of molecularly targeted cancer therapy have promoted efforts to identify systematically the genetic alterations in human cancer. By high-throughput sequencing of tyrosine kinase genes in human non-small-cell lung cancer, we identified somatic mutations in the kinase domain of the epidermal growth factor receptor tyrosine kinase gene (EGFR) that are correlated with clinical response to EGFR tyrosine kinase inhibitors (TKIs). We have shown that these mutant forms of EGFR induce oncogenic transformation in different cellular systems. Cells whose growth depends on EGFR with mutations in exons 19 and 21 are sensitive to EGFR-TKIs, whereas cells expressing insertion mutations in exon 20 or the T790M point mutant, found in tumor biopsies from patients that relapsed after an initial response to EGFR-TKIs, are resistant. Furthermore, by applying a novel, massively parallel sequencing technology, we have shown that clinically relevant oncogene mutations can be detected in clinical specimens with very low tumor content, thereby enabling optimal patient selection for mutation-directed therapy. In summary, by applying high-throughput genomic resequencing, we have identified a novel therapeutic target, mutant EGFR, in lung cancer and evaluated its role in predicting response to targeted therapy.

Whole genome amplification methods are used to generate the large amounts of DNA that are required for genetic testing. Preparation of genomic DNA from clinical samples is a bottleneck in high-throughput genotyping and is frequently limited by the amount of specimen available. Precious DNA collections used for association and linkage analysis can be a nonrenewable resource available to only a limited number of laboratories. To address these needs, amplified DNA is now being used in a growing number of research and diagnostic applications. A new method, called multiple displacement amplification, dramatically improves the high-fidelity reproduction of genomic DNA, with 10–100 kb amplified DNA products providing uniform coverage of genes.

Fundamental to most genetic analysis is availability of genomic DNA of adequate quality and quantity. Because DNA yield from human samples is frequently limiting, much effort has been invested in developing methods for whole genome amplification (WGA) by random or degenerate oligonucleotide-primed PCR. However, existing WGA methods like degenerate oligonucleotide-primed PCR suffer from incomplete coverage and inadequate average DNA size. We describe a method, termed multiple displacement amplification (MDA), which provides a highly uniform representation across the genome. Amplification bias among eight chromosomal loci was less than 3-fold in contrast to 4–6 orders of magnitude for PCR-based WGA methods. Average product length was >10 kb. MDA is an isothermal, strand-displacing amplification yielding about 20–30 μg product from as few as 1–10 copies of human genomic DNA. Amplification can be carried out directly from biological samples including crude whole blood and tissue culture cells. MDA-amplified human DNA is useful for several common methods of genetic analysis, including genotyping of single nucleotide polymorphisms, chromosome painting, Southern blotting and restriction fragment length polymorphism analysis, subcloning, and DNA sequencing. MDA-based WGA is a simple and reliable method that could have significant implications for genetic studies, forensics, diagnostics, and long-term sample storage.

The genetic relatedness of mountain gorillas and humans has led to concerns about interspecies transmission of infectious agents. Human-to-gorilla transmission may explain human metapneumovirus in 2 wild mountain gorillas that died during a respiratory disease outbreak in Rwanda in 2009. Surveillance is needed to ensure survival of these critically endangered animals.

The adaptive immune system confers protection by generating a diverse repertoire of antibody receptors that are rapidly expanded and contracted in response to specific targets. Next-generation DNA sequencing now provides the opportunity to survey this complex and vast repertoire. In the present work, we describe a set of tools for the analysis of antibody repertoires and their application to elucidating the dynamics of the response to viral vaccination in human volunteers. By analyzing data from 38 separate blood samples across 2 y, we found that the use of the germ-line library of V and J segments is conserved between individuals over time. Surprisingly, there appeared to be no correlation between the use level of a particular VJ combination and degree of expansion. We found the antibody RNA repertoire in each volunteer to be highly dynamic, with each individual displaying qualitatively different response dynamics. By using combinatorial phage display, we screened selected VH genes paired with their corresponding VL library for affinity against the vaccine antigens. Altogether, this work presents an additional set of tools for profiling the human antibody repertoire and demonstrates characterization of the fast repertoire dynamics through time in multiple individuals responding to an immune challenge.

The noncoding RNA Xist has been shown to be essential for X-chromosome inactivation and to coat the inactive X-chromosome (Xi). Thus, an important question in understanding the formation of Xi is whether the binding reaction of Xist is necessary for X-chromosome inactivation. In this article, we demonstrate the failure of X-chromosome silencing if the association of Xist with the X-chromosome is inhibited. The chromatin-binding region was functionally mapped and evaluated by using an approach for studying noncoding RNA function in living cells that we call peptide nucleic acid (PNA) interference mapping. In the reported experiments, a single 19-bp antisense cell-permeating PNA targeted against a particular region of Xist RNA caused the disruption of the Xi. The association of the Xi with macro-histone H2A is also disturbed by PNA interference mapping.

We have extended our earlier work to show that individual 14-20mer peptide nucleic acid probes directed against interspersed alpha-satellite sequences can specifically identify chromosomes. Peptide nucleic acid (PNA) probes were used to detect chromosomal abnormalities and repeat structure in the human genome by fluorescence in situ hybridization (FISH). The hybridization of a single PNA probe species directed against a highly abundant alpha-satellite DNA repeat sequence was sufficient to absolutely identify a chromosome. Selection of highly repetitive or region-specific DNA repeats involved DNA database analysis. Distribution of a specific repeat sequence in human genome was estimated through two means: a computer program "whole genome" approach based on approximately 400 Mb (12%) human genomic sequence. The other method involved directed search for alpha satellite sequences. In total, approximately 240 unique DNA repeat candidates were found. Forty-two PNA probes were designed for screening chromosome-specific probes. Ten chromosome-specific PNA probes for human Chromosomes (Chrs) 1, 2, 7, 9, 11, 17, 18, X, and Y have been identified. Interphase and metaphase results demonstrate that chromosome-specific PNA probes are capable of detecting simple aneuploidies (trisomies) in human. Another set of PNA probes showed distinct banding-like patterns and could be used as sequence-specific stains for chromosome "bar coding". Potential application of PNA probes for investigating repeat structure and function is also discussed.

The satellite repeat structure of the mammalian centromere contains the CENP-B protein binding site. Using the peptide nucleic acid-fluorescence in situ hybridization (PNA-FISH), we show by direct PNA-DNA binding that all detectable CENP-B sites in a mammalian genome might have the same sequence. Two species-specific PNA 17-mers, pMm and pMc, were identified from CENP-B binding sites of Mus musculus and M. caroli, respectively. Fluorescence in situ hybridization confirmed that pMc hybridized to M. caroli centromeres only; however, pMm cross-hybridized to M. musculus and human centromeres. By using a series of CENP-B PNA 17-mers containing 1, 2, 3, 5, and 7 base-pair mismatches to their DNA counterparts, we further demonstrate that PNA-FISH can discriminate between two CENP-B DNA sequences that differ by a single base-pair in mouse and human centromeres, suggesting the degree of conservation of CENP-B sequences throughout the genome. In comparison with DNA oligonucleotides, PNA oligomers demonstrate the higher sequence specificity, improved stability, reproducibility, and lower background. Therefore, PNA oligomers have significant advantages over DNA oligonucleotide probes in analyzing microsatellites in a genome.

Peptide nucleic acids (PNAs) can bind to single-stranded DNA by Watson–Crick base pairing and can form triple helices via Hoogsteen bonding to DNA/PNA duplexes. A single dimeric PNA molecule can form a clamp via both double- and triple-helix formation. We designed PNAs to bind as clamps to a site in the supFG1 mutation reporter gene carried within a chromosomally integrated, recoverable λ phage shuttle vector in mouse fibroblasts. The PNAs were introduced into the cells via permeabilization with streptolysin-O, and cellular uptake was confirmed by fluorescein labeling and fluorescent microscopy. PNAs specific for either an 8- or a 10-bp site in the supFG1 gene were found to induce mutations at frequencies in the range of 0.1%, 10-fold above the background. PNAs with three or four mismatches showed poor in vitro target site binding and were ineffective in the mutagenesis assay. No increased mutagenesis was detected with any of the PNAs in the nontargeted cII gene, also carried within the λ vector, further indicating the specificity of the PNA-induced mutagenesis. DNA sequence analysis revealed that the majority of the mutations were located within the PNA-binding site and consisted mostly of single base pair insertions and deletions within the poly G:C run there, suggesting that a high affinity PNA clamp constitutes a mutagenic lesion that may provoke replication slippage errors. The ability to direct mutations to a target site in chromosomal DNA by using PNAs may provide a useful tool for research and therapeutic applications.

Three protocols in this issue highlight applications of emulsification procedures, which deliver high-throughput potential to the molecular biology laboratory, without the need for automation. These procedures have already generated interesting results and spurred the development of exciting new technologies, while requiring only readily available laboratory equipment.

The positively charged and relatively flexible PNA unit ethT (Fig. 1) has an ethylene linker between backbone and nucleobase rather than the methylene carbonyl linker normally present in PNA. The synthesis of the corresponding modified monomer, its incorporation into PNA oligomers, and their hybridization properties to DNA are described.

A peptide nucleic acid (PNA) with improved strand-displacement capability and a site-specific DNA cleavage function is a novel reagent for probing the structure of PNA-DNA complexes in solution. By linking two PNAs in tandem with an aliphatic linker, the bis-PNA forms a bis-PNA-DNA triple-stranded complex having a higher stability to thermal denaturation than conventional monomeric PNAs. When a Gly-Gly-His tripeptide is placed on either the Watson-Crick or Hoogsteen bis-PNA strand, nickel-mediated cleavage is detected at specific sites on the displaced and hybridized DNA strands. Because the displaced strand is cleaved when GGH is placed on either PNA strand, the D-loop must be close to the backbone of the bis-PNA-DNA triplex. Furthermore, the pattern of cleavage on the displaced strand suggests the nickel-tripeptide complex lies in a groove formed by the displaced DNA strand and both PNA strands. These observations suggest that the D-loop is a part of a four-stranded bis-PNA-DNA2 bundle.

The present invention relates to a new functionalized achiral linker reagent for incorporating multiple primary amino groups or reporter groups into oligonucleotides following the phosphoramidite methodology. It is possible to substitute any ribodeoxynucleotide, deoxynucleotide, or nucleotide with the linker in conventional phosphoamidite or H-phosphonate DNA syntheses. Directly, or via a post modification step, an oligonucleotide is labelled with one or more reporter moieties, e.g. dansyl (5-dimethylamino)-1-naphthalenesulfonyl), biotin, digoxigenin, DOXYL (N-oxyl-4,4-dimethyloxazolidine), PROXYL (N-oxyl-2,2,5,5-tetramethylpyrrolidine), TEMPO (N-oxyl-2,2,6,6-tetramethylpiperidine), dinitrophenyl, texas red, tetramethyl rhodamine, 7-nitrobenzo-2-oxa-1-diazole (NBD), or pyrene. The present invention also relates to a solid phase support, e.g. a Controlled Pore Glass (CPG), immobilized linker reagent, to a method for preparing a labelled oligonucleotide, and to the use of the labelled oligonucleotide as hybridisation probe, in polymerase chain reactions (PCR), in nucleic acid sequencing, in cloning recombinant DNA and in vitro mutagenesis.

N -(2-benzyloxyethyl)- N -(thymin-1-ylacetyl)glycine was synthesised and used as a linker for coupling DNA and PNA together. Three different 10-mers composed of 6 DNA units and 4 PNA thymine units were made. The PNA-DNA chimera hybridise to complementary DNA or PNA oligomers, and both the PNA and the DNA part of the chimera are involved in the binding.

Peptide nucleic acids (PNA) were synthesized by a modified Merrifield method using several improvements. Activation by O-[benzotriazol-1-yl]-1,1,3,3-tetramethyluronium hexafluorophosphate in combination with in situ neutralization of the resin allowed efficient coupling of all four Boc-protected PNA monomers within 30 min. HPLC analysis of the crude product obtained from a fully automated synthesis of the model PNA oligomer H-CGGACTAAGTCCATTGC-Gly-NH2, indicated an average yield per synthetic cycle of 97.1%. N1-benzyloxycarbonyl-N6(3)-methylimidazole triflate substantially outperformed acetic anhydride as a capping reagent. The resin-bound PNAs were successfully cleaved by the 'low-high' trifluoromethanesulphonic acid procedure.

The microbial conversion of solid cellulosic biomass to liquid biofuels may provide a renewable energy source for transportation fuels. Endophytes represent a promising group of organisms, as they are a mostly untapped reservoir of metabolic diversity. They are often able to degrade cellulose, and they can produce an extraordinary diversity of metabolites. The filamentous fungal endophyte Ascocoryne sarcoides was shown to produce potential-biofuel metabolites when grown on a cellulose-based medium; however, the genetic pathways needed for this production are unknown and the lack of genetic tools makes traditional reverse genetics difficult. We present the genomic characterization of A. sarcoides and use transcriptomic and metabolomic data to describe the genes involved in cellulose degradation and to provide hypotheses for the biofuel production pathways. In total, almost 80 biosynthetic clusters were identified, including several previously found only in plants. Additionally, many transcriptionally active regions outside of genes showed condition-specific expression, offering more evidence for the role of long non-coding RNA in gene regulation. This is one of the highest quality fungal genomes and, to our knowledge, the only thoroughly annotated and transcriptionally profiled fungal endophyte genome currently available. The analyses and datasets contribute to the study of cellulose degradation and biofuel production and provide the genomic foundation for the study of a model endophyte system., Author Summary A renewable source of energy is a pressing global need. The biological conversion of lignocellulose to biofuels by microorganisms presents a promising avenue, but few organisms have been studied thoroughly enough to develop the genetic tools necessary for rigorous experimentation. The filamentous-fungal endophyte A. sarcoides produces metabolites when grown on a cellulose-based medium that include eight-carbon volatile organic compounds, which are potential biofuel targets. Here we use broadly applicable methods including genomics, transcriptomics, and metabolomics to explore the biofuel production of A. sarcoides. These data were used to assemble the genome into 16 scaffolds, to thoroughly annotate the cellulose-degradation machinery, and to make predictions for the production pathway for the eight-carbon volatiles. Extremely high expression of the gene swollenin when grown on cellulose highlights the importance of accessory proteins in addition to the enzymes that catalyze the breakdown of the polymers. Correlation of the production of the eight-carbon biofuel-like metabolites with the expression of lipoxygenase pathway genes suggests the catabolism of linoleic acid as the mechanism of eight-carbon compound production. This is the first fungal genome to be sequenced in the family Helotiaceae, and A. sarcoides was isolated as an endophyte, making this work also potentially useful in fungal systematics and the study of plant–fungus relationships.

The effects of PNA (peptide nucleic acid) bound to double-stranded (ds) DNA targets positioned downstream from phage T3 or T7 promoters in pBluescriptKS+ derived plasmids on transcription by RNA polymerases T3 or T7 have been studied. The dsDNA targets A10, 5'-A5GA4 or 5'-A2GA2GA4, and the corresponding PNAs T10, T5CT4 and T2CT2CT4 were used and the target-PNA strand displacement complexes were performed in low-salt buffer, since PNA does not bind efficiently to ds DNA in higher salt than 50 mM. It is shown that transcription elongation is arrested at the target site with PNA bound to the template strand, whereas only a marginal effect is observed with PNA bound to the non-template strand. With PNA T10, transcription arrest occurs at the first base of the PNA-binding site, while the arrest with the PNA T5CT4 takes place 2-3 nt inside the PNA binding site. In the case of PNA T2CT2CT4 the arrest is less efficient and occurs at the last 1-3 nt of the binding site. Transcription arrest was also shown for PNAs T6 and T8, although with a much lower efficiency. These results show that efficient transcription elongation arrest can be obtained by PNA targeting of the template DNA strand.

The preparation of mixed-sequence PNAs (PNAs containing the four natural nucleobases; thymine, cytosine, adenine, and guanine) is described. The PNA monomers containing thymine, Cbz-protected cytosine, or adenine or benzyl-protected guanine were prepared via convergent syntheses. Subsequent to introduction of the carboxymethyl linker at N-1 of the pyrimidines or N-9 of the purines and suitable protection of exocyclic groups, the nucleobase derivatives were coupled to the Boc-protected backbone esters 10a or 10b and finally hydrolyzed affording the monomers 12a-d. The exocyclic amino groups of cytosine and adenine were protected with a benzyloxycarbonyl group. The exocyclic amino groups of guanine was left unprotected whereas O 6 was protected as the benzyl ether. Two mixed-sequence 10-mers, each with five purines incorporated, and a mixed-sequence 15-mer containing seven purines were assembled essentially following standard solid phase peptide synthesis protocols

The stability of a new type of DNA mimic, peptide nucleic acid (PNA) in human blood serum, Eschericia coli and Micrococcus luteus extracts and nuclear and cytoplasmic extracts from mouse Ehrlich ascites tumor cells was investigated using HPLC analysis. Under conditions that caused complete cleavage of a control peptide, adrenocorticotropic hormone fragment 4-10, no significant degradation of the PNAs, H-T10-LysNH2 and H-TGTACGTCACAACTA-NH2, could be detected. Similarly, PNA H-T5-LysNH2 was found to resist attack by fungal proteinase K or porcine intestinal mucosa peptidase at concentrations exceeding those necessary to completely degrade a control peptide, H-Phe-Trp-Tyr-Cys-Phe-Trp-Tyr-Lys-Phe-Trp-Tyr-Lys-OH, by at least 1000- and 30-fold, respectively. Thus PNA is expected to have sufficient biostability to be used as a drug.

Peptide nucleic acid (PNA) oligomers where one of the repeating backbone units is extended with a methylene group to either N-(2-aminoethyl)-beta-alanine or N-(3-aminopropyl)glycine were prepared. Alternatively, the linker to the nucleobase was extended from methylenecarbonyl to ethylenecarbonyl. The thermal stability of the hybrids between these PNA oligomers and complementary DNA oligonucleotides was significantly lower than that of the corresponding complexes involving unmodified PNA. However, the sequence selectivity was retained. Thymidyl decamers with all N-(2-aminoethyl)-beta-alanine or N-(3-aminopropyl)glycine backbones were prepared and shown to be unable to hybridize to the complementary (dA)(10) oligonucleotides, whereas a PNA decamer containing only ethylenecarbonyl linkers between the nucleobases and the N-(2-aminoethyl)glycine backbone showed weak but sequence-specific affinity for complementary DNA. All hybrids involving homopyrimidine PNA oligomers exhibited (PNA)(2)/DNA stoichiometry, whereas mixed-sequence PNA oligomers formed PNA/DNA duplexes. The preferred binding direction between the modified PNA and DNA in the duplex motifs was antiparallel, as previously reported for complexes involving unmodified PNA.

A synthesis of N -(2-Boc-aminoethyl)- N -(thymin-1-ylacetyl)alanine ( 5 ) is presented. It is demonstrated that chiral integrity can be preserved. PNA (Peptide Nucleic Acid) containing the D-form of 5 hybridizes to complementary DNA with higher affinity than PNA containing the L-form of 5 .

The vast majority of wine fermentations are performed principally by Saccharomyces cerevisiae. However, there are a growing number of instances in which other species of Saccharomyces play a predominant role. Interestingly, the presence of these other yeast species generally occurs via the formation of interspecific hybrids that contain genomic contributions from both S. cerevisiae and non-S. cerevisiae species. However, despite the large number of wine strains that are characterized at the genomic level, there remains limited information regarding the detailed genomic structure of hybrids used in winemaking. To address this, we describe the genome sequence of the thiol-releasing commercial wine yeast hybrid VIN7. VIN7 is shown to be an almost complete allotriploid interspecific hybrid that is comprised of a heterozygous diploid complement of S. cerevisiae chromosomes and a haploid Saccharomyces kudriavzevii genomic contribution. Both parental strains appear to be of European origin, with the S. cerevisiae parent being closely related to, but distinct from, the commercial wine yeasts QA23 and EC1118. In addition, several instances of chromosomal rearrangement between S. cerevisiae and S. kudriavzevii sequences were observed that may mark the early stages of hybrid genome consolidation. Refereed/Peer-reviewed

Summary Genomic structural variants (SVs) are abundant in humans, differing from other variation classes in extent, origin, and functional impact. Despite progress in SV characterization, the nucleotide resolution architecture of most SVs remains unknown. We constructed a map of unbalanced SVs (i.e., copy number variants) based on whole genome DNA sequencing data from 185 human genomes, integrating evidence from complementary SV discovery approaches with extensive experimental validations. Our map encompassed 22,025 deletions and 6,000 additional SVs, including insertions and tandem duplications. Most SVs (53%) were mapped to nucleotide resolution, which facilitated analyzing their origin and functional impact. We examined numerous whole and partial gene deletions with a genotyping approach and observed a depletion of gene disruptions amongst high frequency deletions. Furthermore, we observed differences in the size spectra of SVs originating from distinct formation mechanisms, and constructed a map constructed a map of SV hotspots formed by common mechanisms. Our analytical framework and SV map serves as a resource for sequencing-based association studies.