A first generation whole genome RH map of the river buffalo with comparison to domestic cattle

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Losing identity: structural diversity of transposable elements belonging to different classes in the genome of Anopheles gambiae

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Searching for molecular markers in head and neck squamous cell carcinomas (HNSCC) by statistical and bioinformatic analysis of larynx-derived SAGE libraries

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Sequenciamento de DNA de nova geração e suas aplicações na genômica de plantas

As plataformas de sequenciamento de nova geração são uma alternativa poderosa para estudos de genômica estrutural e funcional. Na genômica de plantas, os trabalhos com as novas plataformas têm sido destinados ao sequenciamento de transcritos, ressequenciamento ou sequenciamento de novo de genomas plastidiais. Neste trabalho, são detalhadas as tecnologias das plataformas mais utilizadas atualmente, bem como é revisada a aplicação dessas tecnologias na genômica estrutural e funcional de plantas.

Estudos de expressão gênica utilizando-se microarrays: delineamento, análise, e aplicações na pesquisa zootécnica

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Molecular and biochemical characterization of the Neurospora crassa glycogen synthase encoded by the gsn cDNA

Glycogen synthases catalyze the transfer of a glucosyl moiety from a nucleotide phosphosugar to a nascent glycogen chain via an alpha1-->4 linkage. Although many genes coding for glycogen synthases have been described, the enzymes from rabbit and yeast are the best characterized. The fungus Neurospora crassa accumulates glycogen during exponential growth, and mobilizes it at the onset of stationary phase, or when placed at high temperature or starved for carbon. Through a PCR methodology, the gsn cDNA coding for the N. crassa glycogen synthase was isolated, and the amino acid sequence of the protein was deduced. The product of the cDNA seems to be the only glycogen synthase present in N. crassa. Characterization of the gsn cDNA revealed that it codes for a 706-amino acids protein, which is very similar to mammalian and yeast glycogen synthases. Gene expression increased during exponential growth, reaching its maximal level at the end of the exponential growth phase, which is consistent with the pattern of glycogen synthase activity and glycogen level. Expression of the gsn is highly regulated at the transcriptional level. Under culture conditions that induce heat shock, conidiation, and carbon starvation, expression of the gsn gene was decreased, and glycogen synthase activity and glycogen content behaved similarly.

Genomic organization of the Neurospora crassa gsn gene: possible involvement of the STRE and HSE elements in the modulation of transcription during heat shock

Glycogen synthase, an enzyme involved in glycogen biosynthesis, is regulated by phosphorylation and by the allosteric ligand glucose-6-phosphate (G6P). In addition, enzyme levels can be regulated by changes in gene expression. We recently cloned a cDNA for glycogen synthase (gsn) from Neurospora crassa, and showed that gsn transcription decreased when cells were exposed to heat shock (shifted from 30degreesC to 45degreesC). In order to understand the mechanisms that control gsn expression, we isolated the gene, including its 5' and 3' flanking regions, from the genome of N. crassa. An ORF of approximately 2.4 kb was identified, which is interrupted by four small introns (II-V). Intron I (482 bp) is located in the 5'UTR region. Three putative Transcription Initiation Sites (TISs) were mapped, one of which lies downstream of a canonical TATA-box sequence (5'-TGTATAAA-3'). Analysis of the 5'-flanking region revealed the presence of putative transcription factor-binding sites, including Heat Shock Elements (HSEs) and STress Responsive Elements (STREs). The possible involvement of these motifs in the negative regulation of gsn transcription was investigated using Electrophoretic Mobility Shift Assays (EMSA) with nuclear extracts of N. crassa mycelium obtained before and after heat shock, and DNA fragments encompassing HSE and STRE elements from the 5'-flanking region. While elements within the promoter region are involved in transcription under heat shock, elements in the 5'UTR intron may participate in transcription during vegetative growth. The results thus suggest that N. crassa possesses trans-acting elements that interact with the 5'-flanking region to regulate gsn transcription during heat shock and vegetative growth.

DBMODELING - A database applied to the study of protein targets from genome projects

Genome sequencing efforts are providing us with complete genetic blueprints for hundreds of organisms. We are now faced with assigning, understanding, and modifying the functions of proteins encoded by these genomes. DBMODELING is a relational database of annotated comparative protein structure models and their metabolic pathway characterization, when identified. This procedure was applied to complete genomes such as Mycobacteritum tuberculosis and Xylella fastidiosa. The main interest in the study of metabolic pathways is that some of these pathways are not present in humans, which makes them selective targets for drug design, decreasing the impact of drugs in humans. In the database, there are currently 1116 proteins from two genomes. It can be accessed by any researcher at http://www.biocristalografia.df.ibilce.unesp.br/tools/. This project confirms that homology modeling is a useful tool in structural bioinformatics and that it can be very valuable in annotating genome sequence information, contributing to structural and functional genomics, and analyzing protein-ligand docking.

Gene discovery in Boophilus microplus, the cattle tick - the transcriptomes of ovaries, salivary glands, and hemocytes

The quest for new control strategies for ticks can profit from high throughput genomics. In order to identify genes that are involved in oogenesis and development, in defense, and in hematophagy, the transcriptomes of ovaries, hemocytes, and salivary glands from rapidly ingurgitating females, and of salivary glands from males of Boophilus microplus were PCR amplified, and the expressed sequence tags (EST) of random clones were mass sequenced. So far, more than 1,344 EST have been generated for these tissues, with approximately 30% novelty, depending on the the tissue studied. To date approximately 760 nucleotide sequences from B. microplus are deposited in the NCBI database. Mass sequencing of partial cDNAs of parasite genes can build up this scant database and rapidly generate a large quantity of useful information about potential targets for immunobiological or chemical control.

Role of endothelial cell apoptosis in regulation of skeletal muscle angiogenesis during high and low salt intake

Angiogenesis, under normal conditions, is a tightly regulated balance between pro- and antiangiogenic factors. The goal of this study was to investigate the mechanisms involved in the control of the skeletal muscle angiogenic response induced by electrical stimulation during the suppression of plasma renin activity (PRA) with a high-salt diet. Rats fed 0.4% or 4% salt diets were exposed to electrical stimulation for 7 days. The tibialis anterior ( TA) muscles from stimulated and unstimulated hindlimbs were removed and prepared for gene expression analysis, CD31-terminal deoxynucleotide transferase-mediated dUTP nick-end labeling ( TUNEL) double-staining assay, and Bcl-2 and Bax protein expression by Western blot. Rats fed a low-salt diet showed a dramatic angiogenesis response in the stimulated limb compared with the unstimulated limb. This angiogenesis response was significantly attenuated when rats were placed on a high-salt diet. Microarray analysis showed that in the stimulated limb of rats fed a low-salt diet many genes related to angiogenesis were upregulated. In contrast, in rats fed a high-salt diet most of the genes upregulated in the stimulated limb function in apoptosis and cell cycle arrest. Endothelial cell apoptosis, as analyzed by CD31-TUNEL staining, increased by fourfold in the stimulated limb compared with the unstimulated limb. There was also a 48% decrease in the Bcl-2-to-Bax ratio in stimulated compared with unstimulated limbs of rats fed a high-salt diet, confirming severe apoptosis. This study suggests that the increase in endothelial cell apoptosis in TA muscle might contribute to the attenuation of angiogenesis response observed in rats fed a high-salt diet.

Transposable elements in Coffea (Gentianales : Rubiacea) transcripts and their role in the origin of protein diversity in flowering plants

Transposable elements are major components of plant genomes and they influence their evolution, acting as recombination hot spots, acquiring specific cell functions or becoming part of protein-coding regions. The latter is the subject of the present analysis. This study is a report on the annotation of transposable elements (TEs) in expressed sequences of Coffea arabica, Coffea canephora and Coffea racemosa, showing the occurrence of 383 ESTs and 142 unigenes with TE fragments in these three Coffea species. Based on selected unigenes, it was possible to suggest 26 putative proteins with TE-cassette insertions, demonstrating a likely contribution to protein variability. The genes for two of those proteins, the fertility restorer (FR) and the pyrophosphate-dependent phosphofructokinase (PPi-PFKs) genes, were selected for evaluating the impact of TE-cassettes on host gene evolution of other plant genomes (Arabidopsis thaliana, Oryza sativa and populus trichocarpa). This survey allowed identifying a FR gene in O. sativa harboring multiple insertions of LTR retrotransposons that originated new exons, which however does not necessarily mean a case of molecular domestication. A possible transduction event of a fragment of the PPi-PFK beta-subunit gene mediated by Helitron ATREPX1 in Arabidopsis thaliana was also highlighted.

Congenic strains reveal the effect of the renin gene on skeletal muscle angiogenesis induced by electrical stimulation

Previous studies have indicated the importance of angiotensin II (ANG II) in skeletal muscle angiogenesis. The present study explored the effect of regulation of the renin gene on angiogenesis induced by electrical stimulation with the use of physiological, pharmacological, and genetic manipulations of the renin-angiotensin system (RAS). Transfer of the entire chromosome 13, containing the physiologically regulated renin gene, from the normotensive inbred Brown Norway (BN) rat into the background of an inbred substrain of the Dahl salt-sensitive (SS/Mcwi) rat restored renin levels and the angiogenic response after electrical stimulation. This restored response was significantly attenuated when SS-13BN/Mcwi consomic rats were treated with lisinopril or high-salt diet. The role of ANG II on this effect was confirmed by the complete restoration of skeletal muscle angiogenesis in SS/Mcwi rats infused with subpressor doses of ANG II. Congenic strains derived from the SS-13BN/Mcwi consomic were used to further verify the role of the renin gene in this response. Microvessel density was markedly increased after stimulation in congenic strains that contained the renin gene from the BN rat (congenic lines A and D). This angiogenic response was suppressed in control strains that carried regions of the BN genome just above (congenic line C) or just below (congenic line B) the renin gene. The present study emphasizes the importance of maintaining normal renin regulation as well as ANG II levels during the angiogenesis process with a combination of physiological, genetic, and pharmacological manipulation of the RAS.

Vertical inheritance and bursts of transposition have shaped the evolution of the BS non-LTR retrotransposon in Drosophila

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Transcriptome architecture across tissues in the pig

Background: Artificial selection has resulted in animal breeds with extreme phenotypes. As an organism is made up of many different tissues and organs, each with its own genetic programme, it is pertinent to ask: How relevant is tissue in terms of total transcriptome variability? Which are the genes most distinctly expressed between tissues? Does breed or sex equally affect the transcriptome across tissues?Results: In order to gain insight on these issues, we conducted microarray expression profiling of 16 different tissues from four animals of two extreme pig breeds, Large White and Iberian, two males and two females. Mixed model analysis and neighbor - joining trees showed that tissues with similar developmental origin clustered closer than those with different embryonic origins. Often a sound biological interpretation was possible for overrepresented gene ontology categories within differentially expressed genes between groups of tissues. For instance, an excess of nervous system or muscle development genes were found among tissues of ectoderm or mesoderm origins, respectively. Tissue accounted for similar to 11 times more variability than sex or breed. Nevertheless, we were able to confidently identify genes with differential expression across tissues between breeds (33 genes) and between sexes (19 genes). The genes primarily affected by sex were overall different than those affected by breed or tissue. Interaction with tissue can be important for differentially expressed genes between breeds but not so much for genes whose expression differ between sexes.Conclusion: Embryonic development leaves an enduring footprint on the transcriptome. The interaction in gene x tissue for differentially expressed genes between breeds suggests that animal breeding has targeted differentially each tissue's transcriptome.