Physical mapping integrated with syntenic analysis to characterize the gene space of the long arm of wheat chromosome 1A

Background: Bread wheat (Triticum aestivum L.) is one of the most important crops worldwide and its production faces pressing challenges, the solution of which demands genome information. However, the large, highly repetitive hexaploid wheat genome has been considered intractable to standard sequencing approaches. Therefore the International Wheat Genome Sequencing Consortium (IWGSC) proposes to map and sequence the genome on a chromosome-by-chromosome basis. Methodology/Principal Findings: We have constructed a physical map of the long arm of bread wheat chromosome 1A using chromosome-specific BAC libraries by High Information Content Fingerprinting (HICF). Two alternative methods (FPC and LTC) were used to assemble the fingerprints into a high-resolution physical map of the chromosome arm. A total of 365 molecular markers were added to the map, in addition to 1122 putative unique transcripts that were identified by microarray hybridization. The final map consists of 1180 FPC based or 583 LTC based contigs. Conclusions/Significance: The physical map presented here marks an important step forward in mapping of hexaploid bread wheat. The map is orders of magnitude more detailed than previously available maps of this chromosome, and the assignment of over a thousand putative expressed gene sequences to specific map locations will greatly assist future functional studies. This map will be an essential tool for future sequencing of and positional cloning within chromosome 1A

ConservedPrimers 2.0: A high-throughput pipeline for comparative genome referenced intron-flanking PCR primer design and its application in wheat SNP discovery

<p>Abstract</p> <p>Background</p> <p>In some genomic applications it is necessary to design large numbers of PCR primers in exons flanking one or several introns on the basis of orthologous gene sequences in related species. The primer pairs designed by this target gene approach are called "intron-flanking primers" or because they are located in exonic sequences which are usually conserved between related species, "conserved primers". They are useful for large-scale single nucleotide polymorphism (SNP) discovery and marker development, especially in species, such as wheat, for which a large number of ESTs are available but for which genome sequences and intron/exon boundaries are not available. To date, no suitable high-throughput tool is available for this purpose.</p> <p>Results</p> <p>We have developed, the ConservedPrimers 2.0 pipeline, for designing intron-flanking primers for large-scale SNP discovery and marker development, and demonstrated its utility in wheat. This tool uses non-redundant wheat EST sequences, such as wheat contigs and singleton ESTs, and related genomic sequences, such as those of rice, as inputs. It aligns the ESTs to the genomic sequences to identify unique colinear exon blocks and predicts intron lengths. Intron-flanking primers are then designed based on the intron/exon information using the Primer3 core program or BatchPrimer3. Finally, a tab-delimited file containing intron-flanking primer pair sequences and their primer properties is generated for primer ordering and their PCR applications. Using this tool, 1,922 bin-mapped wheat ESTs (31.8% of the 6,045 in total) were found to have unique colinear exon blocks suitable for primer design and 1,821 primer pairs were designed from these single- or low-copy genes for PCR amplification and SNP discovery. With these primers and subsequently designed genome-specific primers, a total of 1,527 loci were found to contain one or more genome-specific SNPs.</p> <p>Conclusion</p> <p>The ConservedPrimers 2.0 pipeline for designing intron-flanking primers was developed and its utility demonstrated. The tool can be used for SNP discovery, genetic variation assays and marker development for any target genome that has abundant ESTs and a related reference genome that has been fully sequenced. The ConservedPrimers 2.0 pipeline has been implemented as a command-line tool as well as a web application. Both versions are freely available at <url>http://wheat.pw.usda.gov/demos/ConservedPrimers/</url>.</p

Expression of p53, inducible nitric oxide synthase and vascular endothelial growth factor in gastric precancerous and cancerous lesions: correlation with clinical features

BACKGROUND: The growth and metastasis of tumors depend on the development of an adequate blood supply via angiogenesis. Recent studies indicate that the inducible nitric oxide synthase (iNOS), vascular endothelial growth factor (VEGF) and the tumor suppressor p53 are fundamental play-markers of the angiogenic process. Overexpression of iNOS and VEGF has been shown to induce angiogenesis in tumors. P53 suppresses angiogenesis by down-regulating VEGF and iNOS. The correlation of expression of p53, VEGF and iNOS and clinical features in gastric carcinogenesis, however, has not been well characterized. METHODS: The expression of p53, iNOS and VEGF in gastric precancerous and cancerous lesions and its relation with the clinical features was determined with immunohistochemistry (avidin-biotin-peroxidase complex method) on 55 randomly selected GC patients and 60 symptom-free subjects from the mass survey in the high-incidence area for GC in Henan, northern China. RESULTS: The positive immunostainig rates for p53, iNOS and VEGF in gastric carcinomas were 51%, 44% and 51%, respectively, and correlated well with TNM stages, but did not show significant difference among the groups with different degrees of gastric wall invasion depth by GC. A positive immunostaining reaction for the iNOS protein was significantly correlated with lymph node metastasis (p = 0.019; Spearman correlation coefficient). P53 protein accumulation was higher in the poorly-differentiated gastric carcinoma than in well-differentiated one. In gastric biopsies, no positive immunosatining was observed for p53, iNOS and VEGF in the histologically normal tissue and chronic superficial gastritis (CSG). However, p53, iNOS and VEGF positive immunostaining was observed in the tissues with different severities of lesions of chronic atrophic gastritis (CAG), intestinal metaplasia (IM) and dysplasia (DYS), and the positive rates increased with the lesion progression from CAG to IM to DYS. A high coincidental positive and negative immunostaining rate for p53, iNOS and VEGF was observed both in biopsy samples with CAG, IM and DYS from the symptom-free subjects and in gastric cancer tissue from the GC patients. CONCLUSIONS: The present results indicated that p53 protein accumulation and increased expression of iNOS and VEGF might be responsible for gastric carcinogenesis and tumor aggressiveness of gastric cancer

Quantum wave mixing and visualisation of coherent and superposed photonic states in a waveguide

Superconducting quantum systems (artificial atoms) have been recently successfully used to demonstrate on-chip effects of quantum optics with single atoms in the microwave range. In particular, a well-known effect of four-wave mixing could reveal a series of features beyond classical physics, when a non-linear medium is scaled down to a single quantum scatterer. Here we demonstrate a phenomenon of the quantum wave mixing (QWM) on a single superconducting artificial atom. In the QWM, the spectrum of elastically scattered radiation is a direct map of the interacting superposed and coherent photonic states. Moreover, the artificial atom visualises photon-state statistics, distinguishing coherent, one- and two-photon superposed states with the finite (quantized) number of peaks in the quantum regime. Our results may give a new insight into nonlinear quantum effects in microwave optics with artificial atoms.Comment: 6 pages, 5 figures; accepted versio

The Metagenome of an Anaerobic Microbial Community Decomposing Poplar Wood Chips

This study describes the composition and metabolic potential of a lignocellulosic biomass degrading community that decays poplar wood chips under anaerobic conditions. We examined the community that developed on poplar biomass in a non-aerated bioreactor over the course of a year, with no microbial inoculation other than the naturally occurring organisms on the woody material. The composition of this community contrasts in important ways with biomass-degrading communities associated with higher organisms, which have evolved over millions of years into a symbiotic relationship. Both mammalian and insect hosts provide partial size reduction, chemical treatments (low or high pH environments), and complex enzymatic ‘secretomes’ that improve microbial access to cell wall polymers. We hypothesized that in order to efficiently degrade coarse untreated biomass, a spontaneously assembled free-living community must both employ alternative strategies, such as enzymatic lignin depolymerization, for accessing hemicellulose and cellulose and have a much broader metabolic potential than host-associated communities. This would suggest that such a community would make a valuable resource for finding new catalytic functions involved in biomass decomposition and gaining new insight into the poorly understood process of anaerobic lignin depolymerization. Therefore, in addition to determining the major players in this community, our work specifically aimed at identifying functions potentially involved in the depolymerization of cellulose, hemicelluloses, and lignin, and to assign specific roles to the prevalent community members in the collaborative process of biomass decomposition. A bacterium similar to Magnetospirillum was identified among the dominant community members, which could play a key role in the anaerobic breakdown of aromatic compounds. We suggest that these compounds are released from the lignin fraction in poplar hardwood during the decay process, which would point to lignin-modification or depolymerization under anaerobic conditions

A Novel Strategy to Construct Yeast Saccharomyces cerevisiae Strains for Very High Gravity Fermentation

Very high gravity (VHG) fermentation is aimed to considerably increase both the fermentation rate and the ethanol concentration, thereby reducing capital costs and the risk of bacterial contamination. This process results in critical issues, such as adverse stress factors (ie., osmotic pressure and ethanol inhibition) and high concentrations of metabolic byproducts which are difficult to overcome by a single breeding method. In the present paper, a novel strategy that combines metabolic engineering and genome shuffling to circumvent these limitations and improve the bioethanol production performance of Saccharomyces cerevisiae strains under VHG conditions was developed. First, in strain Z5, which performed better than other widely used industrial strains, the gene GPD2 encoding glycerol 3-phosphate dehydrogenase was deleted, resulting in a mutant (Z5ΔGPD2) with a lower glycerol yield and poor ethanol productivity. Second, strain Z5ΔGPD2 was subjected to three rounds of genome shuffling to improve its VHG fermentation performance, and the best performing strain SZ3-1 was obtained. Results showed that strain SZ3-1 not only produced less glycerol, but also increased the ethanol yield by up to 8% compared with the parent strain Z5. Further analysis suggested that the improved ethanol yield in strain SZ3-1 was mainly contributed by the enhanced ethanol tolerance of the strain. The differences in ethanol tolerance between strains Z5 and SZ3-1 were closely associated with the cell membrane fatty acid compositions and intracellular trehalose concentrations. Finally, genome rearrangements in the optimized strain were confirmed by karyotype analysis. Hence, a combination of genome shuffling and metabolic engineering is an efficient approach for the rapid improvement of yeast strains for desirable industrial phenotypes