Gramene 2013: comparative plant genomics resources

Gramene (http://www.gramene.org) is a curated online resource for comparative functional genomics in crops and model plant species, currently hosting 27 fully and 10 partially sequenced reference genomes in its build number 38. Its strength derives from the application of a phylogenetic framework for genome comparison and the use of ontologies to integrate structural and functional annotation data. Whole-genome alignments complemented by phylogenetic gene family trees help infer syntenic and orthologous relationships. Genetic variation data, sequences and genome mappings available for 10 species, including Arabidopsis, rice and maize, help infer putative variant effects on genes and transcripts. The pathways section also hosts 10 species-specific metabolic pathways databases developed in-house or by our collaborators using Pathway Tools software, which facilitates searches for pathway, reaction and metabolite annotations, and allows analyses of user-defined expression datasets. Recently, we released a Plant Reactome portal featuring 133 curated rice pathways. This portal will be expanded for Arabidopsis, maize and other plant species. We continue to provide genetic and QTL maps and marker datasets developed by crop researchers. The project provides a unique community platform to support scientific research in plant genomics including studies in evolution, genetics, plant breeding, molecular biology, biochemistry and systems biology

Gramene 2016: comparative plant genomics and pathway resources

Gramene (http://www.gramene.org) is an online resource for comparative functional genomics in crops and model plant species. Its two main frameworks are genomes (collaboration with Ensembl Plants) and pathways (The Plant Reactome and archival BioCyc databases). Since our last NAR update, the database website adopted a new Drupal management platform. The genomes section features 39 fully assembled reference genomes that are integrated using ontology-based annotation and comparative analyses, and accessed through both visual and programmatic interfaces. Additional community data, such as genetic variation, expression and methylation, are also mapped for a subset of genomes. The Plant Reactome pathway portal (http://plantreactome.gramene.org) provides a reference resource for analyzing plant metabolic and regulatory pathways. In addition to approximately 200 curated rice reference pathways, the portal hosts gene homology-based pathway projections for 33 plant species. Both the genome and pathway browsers interface with the EMBL-EBI's Expression Atlas to enable the projection of baseline and differential expression data from curated expression studies in plants. Gramene's archive website (http://archive.gramene.org) continues to provide previously reported resources on comparative maps, markers and QTL. To further aid our users, we have also introduced a live monthly educational webinar series and a Gramene YouTube channel carrying video tutorials

Improvement on the Magnetic Shielding for the XRISM/Resolve Adiabatic Demagnetization Refrigerator

We report modeling, fabrication, cryogenic tensile testing, and magnetic field measurements of a shield around an adiabatic demagnetization stage (ADR) built for the XRISM/Resolve instrument. During testing of a near-identical stage built for Astro-H, a previous spaceflight mission, it was determined that the magnet at full current generated a field external to the shield that violated the maximum dipole-moment requirement of the spacecraft. In addition, there was an interference with the detector assembly nearby when the magnet was greater than 85% of it's typical maximum current. Starting with the Astro-H shield design, we performed a parametric study that increased the thickness of the shield in critical regions. This calculation proceeded until the magnetic field satisfied the estimated maximum field allowed at the detector array based upon the Astro-H measurements. We also performed a detailed measurement of the field generated by the ADR stage at full current as a function of relative angle between the magnet axis and a series of flux-gate magnetometers. Details and results from the calculation and subsequent measurement will be presented

Ensembl Genomes 2013: scaling up access to genome-wide data

Ensembl Genomes (http://www.ensemblgenomes.org) is an integrating resource for genome-scale data from non-vertebrate species. The project exploits and extends technologies for genome annotation, analysis and dissemination, developed in the context of the vertebrate-focused Ensembl project, and provides a complementary set of resources for non-vertebrate species through a consistent set of programmatic and interactive interfaces. These provide access to data including reference sequence, gene models, transcriptional data, polymorphisms and comparative analysis. This article provides an update to the previous publications about the resource, with a focus on recent developments. These include the addition of important new genomes (and related data sets) including crop plants, vectors of human disease and eukaryotic pathogens. In addition, the resource has scaled up its representation of bacterial genomes, and now includes the genomes of over 9000 bacteria. Specific extensions to the web and programmatic interfaces have been developed to support users in navigating these large data sets. Looking forward, analytic tools to allow targeted selection of data for visualization and download are likely to become increasingly important in future as the number of available genomes increases within all domains of life, and some of the challenges faced in representing bacterial data are likely to become commonplace for eukaryotes in future

The Human ABCG1 Transporter Mobilizes Plasma Membrane and Late Endosomal Non-Sphingomyelin-Associated-Cholesterol for Efflux and Esterification

We have previously shown that GFP-tagged human ABCG1 on the plasma membrane (PM) and in late endosomes (LE) mobilizes sterol on both sides of the membrane lipid bilayer, thereby increasing cellular cholesterol efflux to lipid surfaces. In the present study, we examined ABCG1-induced changes in membrane cholesterol distribution, organization, and mobility. ABCG1-GFP expression increased the amount of mobile, non-sphingomyelin(SM)-associated cholesterol at the PM and LE, but not the amount of SM-associated-cholesterol or SM. ABCG1-mobilized non-SM-associated-cholesterol rapidly cycled between the PM and LE and effluxed from the PM to extracellular acceptors, or, relocated to intracellular sites of esterification. ABCG1 increased detergent-soluble pools of PM and LE cholesterol, generated detergent-resistant, non-SM-associated PM cholesterol, and increased resistance to both amphotericin B-induced (cholesterol-mediated) and lysenin-induced (SM-mediated) cytolysis, consistent with altered organization of both PM cholesterol and SM. ABCG1 itself resided in detergent-soluble membrane domains. We propose that PM and LE ABCG1 residing at the phase boundary between ordered (Lo) and disordered (Ld) membrane lipid domains alters SM and cholesterol organization thereby increasing cholesterol flux between Lo and Ld, and hence, the amount of cholesterol available for removal by acceptors on either side of the membrane bilayer for either efflux or esterification

Scission of DNA at a preselected sequence using a single-strand-specific chemical nuclease

Background: We were interested in developing a protocol for cleaving large DNAs specifically. Previous attempts to develop such methods have failed to work because of high levels of nonspecific background scission. Results: R-loop formation was chosen for sequence-specific targeting, a method of hybridization whereby an RNA displaces a DNA strand of identical sequence in 70% formamide using Watson-Crick base-pairing, leading to a three-stranded structure. R-loops are stabilized in aqueous solution by modifying the bases with chemical reagents. The R-loop was cleaved using a novel nuclease prepared from the Thr48→Cys mutant of the single-strand-specific M-13 gene V protein (GVP), which was alkylated with 5-(iodoacetamido-β-alanyl)1,10-phenanthroline. The cleavage products of the pGEM plasmid were cloned into the pCR 2.1-TOPO vector. Adenovirus 2 DNA (35.8 kb; tenfold larger than the pGEM plasmid) was also cleaved quantitatively at a preselected sequence. Conclusions: A new method for cleaving duplex DNA at any preselected sequence was developed. The cleavage method relies on the chemical conversion of M-13 GVP into a nuclease, reflecting GVP's specificity for single-stranded DNA. The GVP chimera is the first example of a semisynthetic secondary structure specific nuclease. The chemical nuclease activity of 1,10-phenanthroline-copper is uniquely suited to this technique because it oxidizes the deoxyribose moiety without generating diffusible intermediates, providing clonable DNA fragments. The protocol could be useful in generating large DNA fragments for mapping the contiguity of probes or defining the exon-intron structure of transcription units