GENOSOJA - The Brazilian Soybean Genome Consortium: high throughput omics and beyond.

Plant genomes are among the most complex and large ones of our planet, with high levels of redundancy when compared to other eukaryotic groups, leading to intricate processes for gene regulation and evolution. Such a complexity demands interdisciplinary and multidimensional approaches in order to allow a better understanding of the processes able to exploit the whole potential of the existing genes in different species, including crop plants. Among cultivated plants, soybean [Glycine max (L.) Merr.] occupies an outstanding position due to its importance as source of protein and oil that may also be converted into biodiesel. The seeds are rarely consumed in natura, but many traditional food products have been consumed, as soymilk, and tofu, as well as fermented products as soy sauce, and soy paste among others, besides its wide use for animal feed. Soybean cultivation has been highly concentrated geographically, with only four countries (USA, Brazil, Argentina and China) accounting for almost 90% of world output. Asia (excluding China) and Africa, the two regions where most of the food insecure countries are located, account for only 5% of production. Among countries classified as 'undernourished', only India and Bolivia are significant producers of soybeans (FAO, 2009). Available evidences indicate that the cultivated soybean was domesticated from its wild relative Glycine soja (Sieb. and Zucc.) in China about 5,000 years ago (Carter et al., 2004). Since then, soybean has been grown primarily in temperate regions for thousands of years, first in Northern Asia and in more recent years in North America and countries of the Southern Cone of Latin America (FAO, 2009). The remarkable success of this crop in temperate zones is well known, but the crop presents also an important role in cropping systems of the tropics and subtropics, also in low fertile regions, as the Brazilian cerrado savannah (Spehar, 1995). The actual area cultivated worldwide with soybean is estimated to cover 103.5 millions of hectares, from which 24.2 only in Brazil, with considerable increases in the production achieved without significant increase in the cultivated area (Embrapa Soybean, 2011). As a legume, soybean is able to develop symbiotic interactions with rhizobia, allowing the fixation and assimilation of atmospheric N2, bearing quite specific mechanisms to coordinate this complex interaction (Oldroyd and Downie, 2008), absent in most angiosperm groups. Besides this peculiarity, soybean presents 2n = 40 chromosomes and was early characterized as an ancient polyploid (paleopolyploid) through genetic mapping studies that identified homeologous chromosome regions based upon duplicate RFLP markers (Shoemaker et al., 1996; Lee et al., 1999; 2001). Due to its allopolyploid nature, the first approaches regarded the generation of expressed sequences from different library tissues and conditions, including mainly ESTs (Expressed Sequence Tags; Nelson et al., 2005) partially in annotated databases, including ca. 40.000 full length cDNA clones available (Umezawa et al., 2008, see also RIKEN Soybean Full-Length cDNA Database), besides analyses regarding RNAseq under different tissues and development stages, as well as under different stressing situations (e.g. Libault et al., 2010; Severin et al., 2010). Also a complete shotgun genome sequence of the soybean cultivar Williams 82 was released, comprising 1.1-gigabase genome size allowing the integration of physical and high-density genetic maps, including 46,430 predicted protein-coding genes (Schmutz et al., 2010). The total amount of data publicly available at GenBank (NCBI) includes more than 120,000 nucleotide sequences (mainly mRNA), ~1,460,000 ESTs, ~368,000 genome sequences, ~80,000 proteins, 118 deposited structures and more than 6,2 million SNPs. Such numbers show that working with soybean is a very challenging task. By the other hand, despite of the wide data availability, most data regard cultivars from temperate regions (as Williams 82), not adapted for cultivation under tropical conditions, as it is the case of central Brazil and many other tropical countries that are subjected to distinct environmental stresses. The proposition of creating the GENOSOJA consortium was submitted in 2007 to the National Council for Scientific and Technological Development (CNPq), an agency linked to the Brazilian Ministry of Science and Technology (MCT), starting its activity in March 2008 with the participation of nine Brazilian institutions from different regions (Figure 1). The proposal aimed to study the soybean genome from its organization into the structural level, seeking to characterize and sequence important genomic regions and their products, thus contributing to the identification of genes using transcriptional and proteomic methods, especially considering the plant response to different biotic and abiotic stresses that affect the culture in the Southern hemisphere. Still, the GENOSOJA network aimed to approach not only whether a gene is induced or suppressed under a given condition, but also to determine the levels at which it is expressed, the interactions with other genes, their physical location and products, allowing the identification of important genes and metabolic pathways, vital for the development and study of plants tolerant to challenging situations. The GENOSOJA project is still in course and is structured into six Project Components (Figure 2), including management and addressing of different aspects of the soybean genome: I. Project management - responsible for the project administration, organization of meetings, group integration and research reports, among others. II. Structural Genomics - includes research activities related to the genomic physical architecture, including BAC anchoring (in the cultivar Conquista), promoter analysis and sequencing of gene-rich regions, also in comparison with other wild relatives of the genus Glycine, allowing studies of synteny and indication of regions important for ressequencing. This component is also responsible for the identification of single base polymorphisms (SNPs), very important for mapping purposes and marker assisted selection. III. Transcriptomics -comprises the largest research group, responsible for various expression profiling approaches using different strategies to access transcripts generated under different biotic (Asian rust: Phakopsora pachyrhizi, CPMMV: Cowpea mild mottle virus, nematodes: Meloydogyne javanica and Pratylenchus brachyurus) and abiotic (water deficit) stresses. In this workgroup different strategies were used, including: a) Subtractive cDNA libraries (76 bp tags, Solexa Illumina® sequencing) using contrasting materials submitted to biotic interactions, including diseases (~40 million tags; Asian rust and virus inoculation) and beneficial interactions (~10 million tags; inoculation with Bradyrhizobium japonicum), as well as water deficit (~42 million tags, comparing tolerant and susceptible accessions). b) SuperSAGE comprising ~3,2 Solexa Illumina® 26-bp tags distributed in six libraries generated under biotic (water deficit) and abiotic (Asian rust) stress comparisons. c) MicroRNA libraries (Solexa Illumina®, 1924 bp) including four libraries regarding water deficit ( 4,8 millions miRNAs) and other four regarding Asian rust (~7,9 million miRNAs). d) cDNA sequences (2,112 sequences, Sanger method) from roots infested with the nematode M. javanica compared with non stressed control. The three first above mentioned experiments were carried out using the same experimental conditions, generating an extensive comparable dataset to allow the understanding of the gene expression dynamic (Subtractive cDNA and SuperSAGE libraries), including biotic and abiotic cross-talk responses as well as the post transcriptional control (miRNA). IV. Proteomics -aimed to study the protein profile of soybean plants, low-mass protein and peptides identification and protein-protein interactions, using the same accessions and biological conditions established for the transcriptomic analyses to ensure complementarity and reduction of experimental variability, and thus, allowing the integration of both datasets in the functional characterization of the soybean genome. V. Expression Assays (transgenesis) -considering the results of transcriptomics and proteomics, most valuable gene candidates are being transformed in order to infer about their effects or biological function. Members of this group are also evaluating the vicinity of genes (UTRs) for the identification of regulatory regions (promoters, enhancers, cis-elements, etc.) that control their expression. VI. Bioinformatics -this workgroup developed the GENOSOJA database (see web resource) that includes a set of tools integrating the entire project data as compared with available sequences from other public data banks. The present issue represents the starting point of an extensive catalogue of products generated by the GENO-SOJA consortium, since all members agree that many additional inferences will be soon mature for publication and application to breeding projects. Thousands of candidate genes differentially expressed have been identified and are being validated using quantitative real time PCR, many regarding strongly induced genes in contrasting libraries (e.g. stressed against control or tolerant against sensible in the same condition). Many of them refer to uncharacterized genes, with no given function, representing relevant data to be worked out in future functional studies, since they may represent not yet described genes, some possibly unique to legumes and important for plant breeding. Finally, the present volume does not represent a milestone for completion of the GENOSOJA project, but an announcement of its birth, crowned with solid growth, integration and consolidation prospects. The data generated by the GENOSOJA consortium will also join the worldwide effort to study the soybean genome through the participation in the International Soybean Genome Consortium (ISGC). In this sense, the next step involves the public release of the generated data, which shall be available for the world community, allowing the effective integration with other networks throughout the world

Hepatic Cytochrome P-450. A Proton Magnetic Relaxation Study of Microsomal, Solubilized and Partially Reconstituted Enzyme System

The longitudiJ:ial proton magnetic relaxation times, Ti, were measured from -5 to 40 °c for microsomal, solubilized and reconstituted cytochrome P-450 obtained from phenobarbital-induced rat livers. The paramagnetic contribution to the rates was derived by subtraction of the rates measured on dithionite-CO-reduced samples. The same values were obtained for microsomal P-450 on reduction with NADPH. PMR titratio.n by KCN yielded a dissociation constant of about 30 mM. This is three orders of magnitude larger than for metmyoglobin. It is concluded that the measured PMR rates are most likely due to the P-450 (and P-420) haem-iron while the 300/o non-haem iron found in both the microsomal and s olubilized P-450 is .ineffective for the PMR rates. These rates increase several times on isotopic dilution (D20 for H20) with the microsomes and diminish for the solubilized samples. Microsomes show 170/o residual, encaged, H20. Most of their paramagnetic PMR rate is due to the parama.gnetic iron located on the outside of microsomes. This is demonstrated by measurements with deuterated samples to which 190/o H20 had been added. Hence, the solubilized P-450 is homogeneous regarding PMR, but the microsomes are not

Hepatic Cytochrome P-450. A Proton Magnetic Relaxation Study of Microsomal, Solubilized and Partially Reconstituted Enzyme System

The longitudiJ:ial proton magnetic relaxation times, Ti, were measured from -5 to 40 °c for microsomal, solubilized and reconstituted cytochrome P-450 obtained from phenobarbital-induced rat livers. The paramagnetic contribution to the rates was derived by subtraction of the rates measured on dithionite-CO-reduced samples. The same values were obtained for microsomal P-450 on reduction with NADPH. PMR titratio.n by KCN yielded a dissociation constant of about 30 mM. This is three orders of magnitude larger than for metmyoglobin. It is concluded that the measured PMR rates are most likely due to the P-450 (and P-420) haem-iron while the 300/o non-haem iron found in both the microsomal and s olubilized P-450 is .ineffective for the PMR rates. These rates increase several times on isotopic dilution (D20 for H20) with the microsomes and diminish for the solubilized samples. Microsomes show 170/o residual, encaged, H20. Most of their paramagnetic PMR rate is due to the parama.gnetic iron located on the outside of microsomes. This is demonstrated by measurements with deuterated samples to which 190/o H20 had been added. Hence, the solubilized P-450 is homogeneous regarding PMR, but the microsomes are not

Solvent Proton Magnetic Relaxation in Solution of Rabbit Liver Cytochrome P450. On the Corfelation Time for the Electron Proton Dipole-Dipole Interaction

Structural parameters can be derived from PMR measurements if the correlation time fdr the spin interactions is known. The relaxation rates induced by the ferric haem-iron of cyt P450 were found to be frequency independent from 10 to 37 MHz. The possible limits for the correlation time according to Solomon\u27s theory are discussed with regard to the possible existence of a water molecule at the sixth coordination site of the haem-iron. No definite conclusion could be reached in this respect, but the results are definetely in favour of a haem environment which can accomodate several water molecules exchanging quickly with the bulk of solvent

Shutdown of achaete-scute homolog-1 expression by heterogeneous nuclear ribonucleoprotein (hnRNP)-A2/B1 in hypoxia

The basic helix-loop-helix transcription factor hASH1, encoded by the ASCL1 gene, plays an important role in neurogenesis and tumor development. Recent findings indicate that the local oxygen tension is a critical determinant for the progression of neuroblastomas. Here we investigated the molecular mechanisms underlying the oxygen-dependent expression of hASH1 in neuroblastoma cells. Exposure of human neuroblastoma-derived Kelly cells to 1% O2 significantly decreased ASCL1 mRNA and hASH1 protein levels. Using reporter gene assays, we show that the response of hASH1 to hypoxia is mediated mainly by post-transcriptional inhibition via the ASCL1 mRNA 5'- and 3'-UTRs, while additional inhibition of the ASCL1 promoter was observed under prolonged hypoxia. By RNA pull-down experiments followed by MALDI/TOF-MS analysis, we identified heterogeneous nuclear ribonucleoprotein (hnRNP)-A2/B1 and hnRNP-R as interactors binding directly to the ASCL1 mRNA 5'- and 3'-UTRs and influencing its expression. We further demonstrate that hnRNP-A2/B1 is a key positive regulator of ASCL1, findings that were also confirmed by analysis of a large compilation of gene expression data. Our data suggest that a prominent down-regulation of hnRNP-A2/B1 during hypoxia is associated with the post-transcriptional suppression of hASH1 synthesis. This novel post-transcriptional mechanism for regulating hASH1 levels will have important implications in neural cell fate development and disease

Distribuição de microssatélites no genoma de leguminosas mediante hibridização in situ fluorescente.

Os microssatélites consistem em unidades de repetição (1 a 5pb) distribuídas em tandem, que podem estar dispersas ao longo dos genomas ou associadas a regiões heterocromáticas. Tais características têm permitido sua utilização na construção de mapas cromossômicos mediante hibridização in situ fluorescente (FISH), auxiliando no entendimento da organização genômica entre espécies proximamente relacionadas. O presente trabalho caracterizou a distribuição de oligonucleotídeos sintéticos (AG)8, (AAG)5, (ACC)5, (CTC)5 e (TGA)6 em Phaseolus vulgaris, P. lunatus, Vigna unguiculata, V. radiata e Glycine max, identificando marcas cromossômicas para a comparação destas leguminosas. Lâminas foram hibridizadas com sondas de oligonucleotídeos e rehibridizadas com DNAr 5S e 45S, provenientes de Lotus japonicus e Arabidopsis thaliana, respectivamente. Em soja, esta análise estendeu-se a uma investigação genômica, a partir de sequências disponibilizadas no banco de dados SoyBase (http://www.soybase.org/), usando o programa BLASTN com os seguintes parâmetros: formato de saída de alinhamentos sem lacunas, matriz de comparação blossum62, valor W padrão, e-value 0.1 ou menor e filtro de baixa complexidade desligado. Os oligonucleotídeos [(AAG)5]10, [(AAC)5]10, [(AG)8]15, [(CTC)5]10 e [(TGA)6]10 foram utilizados como sondas, adotando como ponto de corte valores de identidade de pareamento inferiores a 77%, objetivando caracterizar número, tamanho e localização destas unidades de repetições no genoma. A FISH com oligonucleotídeos evidenciaram marcações pericentroméricas e proximais, embora um padrão de distribuição disperso tenha prevalecido ao longo dos cromossomos das espécies analisadas, com exceção de (AG)8 em soja que não revelou marcações visíveis. O oligonucleotídeo (AAG)5 revelou uma marcação pericentromérica evidente em P. lunatus, tratando-se de um marcador cromossômico para tal espécie. Na hibridização com (ACC)5 notou-se a presença de seis marcações fortes em V. unguiculata, estando uma delas localizada no par cromossômico 10, portador do sítio de DNAr 45S. O microssatélite (TGA)6 revelou uma marcação bem evidente no cromossomo 10 de P. lunatus, identificado pela presença de DNAr 5S. Algumas divergências observadas no padrão de distribuição e na intensidade dos sinais entre os genomas das espécies confirmam a hipótese de que as sequências de DNA repetitivo apresentam uma composição heterogênea. Na análise genômica de soja, foram observados 92, 84, 83, 142 e 103 sítios de repetições para os oligonucleotídeos (AG)8, (AAG)5, (ACC)5, (CTC)5 e (TGA)6, respectivamente, de tamanhos variáveis entre 30 a 454 pb, localizados, principalmente, em regiões de alta a moderada densidade gênica, por vezes associados a genes e elementos transponíveis. Considerando o pequeno tamanho, fica evidente que estas sequências não correspondem aos sítios observados pela FISH, uma vez que esta técnica evidencia apenas sequências com comprimentos acima de 1-3kb. Sugere-se que as marcações localizadas por FISH estejam associadas a regiões de heterocromatina e que sejam constituintes dos mais de 1.000 scaffolds existentes no sequenciamento da soja, não sendo, por essa razão, detectadas pela análise in silico

A new, potent poly(ADP-ribose) polymerase inhibitor improves cardiac and vascular dysfunction associated with advanced aging

Increased production of reactive oxygen and nitrogen species has recently been implicated in the pathogenesis of cardiac and endothelial dysfunction associated with atherosclerosis, hypertension, and aging. Oxidant-induced cell injury triggers the activation of nuclear enzyme poly(ADP-ribose) polymerase (PARP), which in turn contributes to cardiac and vascular dysfunction in various pathophysiological conditions including diabetes, reperfusion injury, circulatory shock, and aging. Here, we investigated the effect of a new PARP inhibitor, INO-1001, on cardiac and endothelial dysfunction associated with advanced aging using Millar's new Aria pressure-volume conductance system and isolated aortic rings. Young adult (3 months old) and aging (24 months old) Fischer rats were treated for 2 months with vehicle, or the potent PARP inhibitor INO-1001. In the vehicle-treated aging animals, there was a marked reduction of both systolic and diastolic cardiac function and loss of endothelial relaxant responsiveness of aortic rings to acetylcholine. Treatment with INO-1001 improved cardiac performance in aging animals and also acetylcholine-induced, nitric oxide-mediated vascular relaxation. Thus, pharmacological inhibition of PARP may represent a novel approach to improve cardiac and vascular dysfunction associated with aging

Exile Vol. XXXII No. 2

ARTWORK Manhole by Linda Gates (cover) Escape by Linda Gates 3 Spring by Aimee Creelman 11 Children on Bridge by Holland Behrens 19 Homestead Instead by Allison Lange 29 Infrared Exploration by Allison Lange 37 Seasons I by Aimee Creelman 47 FICTION My Mother Wears Yellow on Tuesdays by Joan R. DeWitt 5-10 Tilly by Theresa Copeland 21-28 The Rights of Spring by Leigh Walton 40-46 POETRY Learning to Knock by Amy Becker 1 Syndrome by Jeff Masten 2 Beauty and the Beasts by Leigh Walton 13 The Sound and the Silence by Teresa Woodward 14-18 The Dark by Amy Becker 31 By the Toussaint River by Debra Benko 32-33 Wish Dolls by Carrie Jordan 34-35 Bob\u27s Mind Wanders in Class by Amy Becker 36 The Woman I Call Mother by Karen J. Hall 39 CONTRIBUTOR NOTES 49 Editors share equally all editorial decisions In honor of Mr. Paul Bennett, poet and founder of the writing program at Denison, of which EXILE is an expression