Duplex stability of DNA.DNA and DNA.RNA duplexes containing 3 '-S-phosphorothiolate linkages

3′-S-Phosphorothiolate (3′-SP) linkages have been incorporated into the DNA strand of both a DNA·RNA duplex and a DNA·DNA duplex. Thermal melting (Tm) studies established that this modification significantly stabilises the DNA·RNA duplex with an average increase in Tm of about 1.4 °C per modification. For two or three modifications, the increase in Tm was larger for an alternating, as compared to the contiguous, arrangement. For more than three modifications their arrangement had no effect on Tm. In contrast to the DNA·RNA duplex, the 3′-S-phosphorothiolate linkage destabilised the DNA·DNA duplex, irrespective of the arrangement of the 3′-SP linkages. The effect of ionic strength on duplex stability was similar for both the phosphorothiolate-substituted and the unmodified RNA·DNA duplexes. The results are discussed in terms of the influence that the sulfur atom has on the conformation of the furanose ring and comparisons are also drawn between the current study and those previously conducted with other modifications that have a similar conformational effect.

Synthesis of 3 '-S-phosphorothiolate oligonucleotides for their potential use in RNA interference

The potency of RNA interference (RNAi) undoubtedly can be improved through chemical modifications to the small interfering RNAs (siRNA). By incorporation of the 3′-S-phosphorothiolate modification into strands of RNA, it is hoped that specific regions of a siRNA duplex can be stabilised to enhance the target binding affinity of a selected antisense strand into the activated RNA-induced silencing complex (RISC*). Oligonucleotides composed entirely of this modification are desirable so unconventional 5′ → 3′ synthesis is investigated, with initial solution-phase testing proving successful. The phosphoroamidite monomer required for solid-phase synthesis has also been produced.

Low relative humidity triggers RNA-directed de novo DNA methylation and suppression of genes controlling stomatal development

Environmental cues influence the development of stomata on the leaf epidermis, and allow plants to exert plasticity in leaf stomatal abundance in response to the prevailing growing conditions. It is reported that Arabidopsis thaliana ‘Landsberg erecta’ plants grown under low relative humidity have a reduced stomatal index and that two genes in the stomatal development pathway, SPEECHLESS and FAMA, become de novo cytosine methylated and transcriptionally repressed. These environmentally-induced epigenetic responses were abolished in mutants lacking the capacity for de novo DNA methylation, for the maintenance of CG methylation, and in mutants for the production of short-interfering non-coding RNAs (siRNAs) in the RNA-directed DNA methylation pathway. Induction of methylation was quantitatively related to the induction of local siRNAs under low relative humidity. Our results indicate the involvement of both transcriptional and post-transcriptional gene suppression at these loci in response to environmental stress. Thus, in a physiologically important pathway, a targeted epigenetic response to a specific environmental stress is reported and several of its molecular, mechanistic components are described, providing a tractable platform for future epigenetics experiments. Our findings suggest epigenetic regulation of stomatal development that allows for anatomical and phenotypic plasticity, and may help to explain at least some of the plant’s resilience to fluctuating relative humidity.

Cell walls of developing wheat starchy endosperm: comparison of composition and RNA-Seq transcriptome

The transcriptome of the developing starchy endosperm of hexaploid wheat (Triticum aestivum) was determined using RNA-Seq isolated at five stages during grain fill. This resource represents an excellent way to identify candidate genes responsible for the starchy endosperm cell wall, which is dominated by arabinoxylan (AX), accounting for 70% of the cell wall polysaccharides, with 20% (1,3; 1,4)-beta-D-glucan, 7% glucomannan, and 4% cellulose. A complete inventory of transcripts of 124 glycosyltransferase (GT) and 72 glycosylhydrolase (GH) genes associated with cell walls is presented. The most highly expressed GT transcript (excluding those known to be involved in starch synthesis) was a GT47 family transcript similar to Arabidopsis (Arabidopsis thaliana) IRX10 involved in xylan extension, and the second most abundant was a GT61. Profiles for GT43 IRX9 and IRX14 putative orthologs were consistent with roles in AX synthesis. Low abundances were found for transcripts from genes in the acyl-coA transferase BAHD family, for which a role in AX feruloylation has been postulated. The relative expression of these was much greater in whole grain compared with starchy endosperm, correlating with the levels of bound ferulate. Transcripts associated with callose (GSL), cellulose (CESA), pectin (GAUT), and glucomannan (CSLA) synthesis were also abundant in starchy endosperm, while the corresponding cell wall polysaccharides were confirmed as low abundance (glucomannan and callose) or undetectable (pectin) in these samples. Abundant transcripts from GH families associated with the hydrolysis of these polysaccharides were also present, suggesting that they may be rapidly turned over. Abundant transcripts in the GT31 family may be responsible for the addition of Gal residues to arabinogalactan peptide.

Partial identification of spirochaetes from two dairy cows with digital dermatitis by polymerase chain reaction analysis of the 16S ribosomal RNA gene

Specimens taken postmortem from typical lesions of digital dermatitis in two dairy cows were tested by the polymerase chain reaction (PCR) for the presence of a spirochaetal 16S rRNA gene. Seven different assays detected the gene in the samples from both cows. Two of the PCR products were sequenced and a comparison of the nucleotide sequences revealed that the spirochaete belonged to the genus Treponema and was closely related to Treponema denticola. A PCR specific for the detection of the digital dermatitis-associated treponeme was developed.

RNA interference suppression of genes in glycosyl transferase families 43 and 47 in wheat starchy endosperm causes large decreases in arabinoxylan content

The cell walls of wheat (Triticum aestivum) starchy endosperm are dominated by arabinoxylan (AX), accounting for 65% to 70% of the polysaccharide content. Genes within two glycosyl transferase (GT) families, GT43 (IRREGULAR XYLEM9 [IRX9] and IRX14) and GT47 (IRX10), have previously been shown to be involved in the synthesis of the xylan backbone in Arabidopsis, and close homologs of these have been implicated in the synthesis of xylan in other species. Here, homologs of IRX10 TaGT47_2 and IRX9 TaGT43_2, which are highly expressed in wheat starchy endosperm cells, were suppressed by RNA interference (RNAi) constructs driven by a starchy endosperm-specific promoter. The total amount of AX was decreased by 40% to 50% and the degree of arabinosylation was increased by 25% to 30% in transgenic lines carrying either of the transgenes. The cell walls of starchy endosperm in sections of grain from TaGT43_2 and TaGT47_2 RNAi transgenics showed decreased immunolabeling for xylan and arabinoxylan epitopes and approximately 50% decreased cell wall thickness compared with controls. The proportion of AX that was water soluble was not significantly affected, but average AX polymer chain length was decreased in both TaGT43_2 and TaGT47_2 RNAi transgenics. However, the long AX chains seen in controls were absent in TaGT43_2 RNAi transgenics but still present in TaGT47_2 RNAi transgenics. The results support an emerging picture of IRX9-like and IRX10-like proteins acting as key components in the xylan synthesis machinery in both dicots and grasses. Since AX is the main component of dietary fiber in wheat foods, the TaGT43_2 and TaGT47_2 genes are of major importance to human nutrition.

How the double spherules of infectious bronchitis virus impact our understanding of RNA virus replicative organelles

Powered by advances in electron tomography, recent studies have extended our understanding of how viruses construct "replication factories" inside infected cells. Their function, however, remains an area of speculation with important implications for human health. It is clear from these studies that whatever their purpose, organelle structure is dynamic (M. Ulasli, M. H. Verheije, C. A. de Haan, and F. Reggiori, Cell. Microbiol. 12:844-861, 2010) and intricate (K. Knoops, M. Kikkert, S. H. Worm, J. C. Zevenhoven-Dobbe, Y. van der Meer, et al., PLOS Biol. 6:e226, 2008). But by concentrating on medically important viruses, these studies have failed to take advantage of the genetic variation inherent in a family of viruses that is as diverse as the archaea, bacteria, and eukaryotes combined (C. Lauber, J. J. Goeman, M. del Carmen Parquet, P. T. Nga, E. J. Snijder, et al., PLOS Pathog. 9:e1003500, 2013). In this climate, Maier et al. (H. J. Maier, P. C. Hawes, E. M. Cottam, J. Mantell, P. Verkade, et al., mBio 4:e00801-13, 2013) explored the replicative structures formed by an avian coronavirus that appears to have diverged at an early point in coronavirus evolution and shed light on controversial aspects of viral biology.

Ecuador Paraiso Escondido virus, a new flavivirus isolated from New World sand flies in Ecuador, is the first representative of a novel clade in the genus flavivirus

A new flavivirus, Ecuador Paraiso Escondido virus (EPEV), named after the village where it was discovered, was isolated from sand flies (Psathyromyia abonnenci, formerly Lutzomyia abonnenci) that are unique to the New World. This represents the first sand fly-borne flavivirus identified in the New World. EPEV exhibited a typical flavivirus genome organization. Nevertheless, the maximum pairwise amino acid sequence identity with currently recognized flaviviruses was 52.8%. Phylogenetic analysis of the complete coding sequence showed that EPEV represents a distinct clade which diverged from a lineage that was ancestral to the nonvectored flaviviruses Entebbe bat virus, Yokose virus, and Sokoluk virus and also the Aedes-associated mosquito-borne flaviviruses, which include yellow fever virus, Sepik virus, Saboya virus, and others. EPEV replicated in C6/36 mosquito cells, yielding high infectious titers, but failed to reproduce either in vertebrate cell lines (Vero, BHK, SW13, and XTC cells) or in suckling mouse brains. This surprising result, which appears to eliminate an association with vertebrate hosts in the life cycle of EPEV, is discussed in the context of the evolutionary origins of EPEV in the New World.The flaviviruses are rarely (if ever) vectored by sand fly species, at least in the Old World. We have identified the first representative of a sand fly-associated flavivirus, Ecuador Paraiso Escondido virus (EPEV), in the New World. EPEV constitutes a novel clade according to current knowledge of the flaviviruses. Phylogenetic analysis of the virus genome showed that EPEV roots the Aedes-associated mosquito-borne flaviviruses, including yellow fever virus. In light of this new discovery, the New World origin of EPEV is discussed together with that of the other flaviviruses.