Fourmidable: a database for ant genomics.

BACKGROUND: Fourmidable is an infrastructure to curate and share the emerging genetic, molecular, and functional genomic data and protocols for ants. DESCRIPTION: The Fourmidable assembly pipeline groups nucleotide sequences into clusters before independently assembling each cluster. Subsequently, assembled sequences are annotated via Interproscan and BLAST against general and insect-specific databases. Gene-specific information can be retrieved using gene identifiers, searching for similar sequences or browsing through inferred Gene Ontology annotations. The database will readily scale as ultra-high throughput sequence data and sequences from additional species become available. CONCLUSION: Fourmidable currently houses EST data from two ant species and microarray gene expression data for one of these. Fourmidable is publicly available at http://fourmidable.unil.ch.

CATMA: a complete Arabidopsis GST database.

The Complete Arabidopsis Transcriptome Micro Array (CATMA) database contains gene sequence tag (GST) and gene model sequences for over 70% of the predicted genes in the Arabidopsis thaliana genome as well as primer sequences for GST amplification and a wide range of supplementary information. All CATMA GST sequences are specific to the gene for which they were designed, and all gene models were predicted from a complete reannotation of the genome using uniform parameters. The database is searchable by sequence name, sequence homology or direct SQL query, and is available through the CATMA website at http://www.catma.org/.

Density-based hierarchical clustering of pyro-sequences on a large scale--the case of fungal ITS1.

MOTIVATION: Analysis of millions of pyro-sequences is currently playing a crucial role in the advance of environmental microbiology. Taxonomy-independent, i.e. unsupervised, clustering of these sequences is essential for the definition of Operational Taxonomic Units. For this application, reproducibility and robustness should be the most sought after qualities, but have thus far largely been overlooked. RESULTS: More than 1 million hyper-variable internal transcribed spacer 1 (ITS1) sequences of fungal origin have been analyzed. The ITS1 sequences were first properly extracted from 454 reads using generalized profiles. Then, otupipe, cd-hit-454, ESPRIT-Tree and DBC454, a new algorithm presented here, were used to analyze the sequences. A numerical assay was developed to measure the reproducibility and robustness of these algorithms. DBC454 was the most robust, closely followed by ESPRIT-Tree. DBC454 features density-based hierarchical clustering, which complements the other methods by providing insights into the structure of the data. AVAILABILITY: An executable is freely available for non-commercial users at ftp://ftp.vital-it.ch/tools/dbc454. It is designed to run under MPI on a cluster of 64-bit Linux machines running Red Hat 4.x, or on a multi-core OSX system. CONTACT: dbc454@vital-it.ch or nicolas.guex@isb-sib.ch.

Similarities and differences of polyadenylation signals in human and fly.

BACKGROUND: Cleavage of messenger RNA (mRNA) precursors is an essential step in mRNA maturation. The signal recognized by the cleavage enzyme complex has been characterized as an A rich region upstream of the cleavage site containing a motif with consensus AAUAAA, followed by a U or UG rich region downstream of the cleavage site. RESULTS: We studied these signals using exhaustive databases of cleavage sites obtained from aligning raw expressed sequence tags (EST) sequences to genomic sequences in Homo sapiens and Drosophila melanogaster. These data show that the polyadenylation signal is highly conserved in human and fly. In addition, de novo motif searches generated a refined description of the U-rich downstream sequence (DSE) element, which shows more divergence between the two species. These refined motifs are applied, within a Hidden Markov Model (HMM) framework, to predict mRNA cleavage sites. CONCLUSION: We demonstrate that the DSE is a specific motif in both human and Drosophila. These findings shed light on the sequence correlates of a highly conserved biological process, and improve in silico prediction of 3' mRNA cleavage and polyadenylation sites.

Integrated methodologies to study human transcriptional regulation from functional genomics data

Abstract : The human body is composed of a huge number of cells acting together in a concerted manner. The current understanding is that proteins perform most of the necessary activities in keeping a cell alive. The DNA, on the other hand, stores the information on how to produce the different proteins in the genome. Regulating gene transcription is the first important step that can thus affect the life of a cell, modify its functions and its responses to the environment. Regulation is a complex operation that involves specialized proteins, the transcription factors. Transcription factors (TFs) can bind to DNA and activate the processes leading to the expression of genes into new proteins. Errors in this process may lead to diseases. In particular, some transcription factors have been associated with a lethal pathological state, commonly known as cancer, associated with uncontrolled cellular proliferation, invasiveness of healthy tissues and abnormal responses to stimuli. Understanding cancer-related regulatory programs is a difficult task, often involving several TFs interacting together and influencing each other's activity. This Thesis presents new computational methodologies to study gene regulation. In addition we present applications of our methods to the understanding of cancer-related regulatory programs. The understanding of transcriptional regulation is a major challenge. We address this difficult question combining computational approaches with large collections of heterogeneous experimental data. In detail, we design signal processing tools to recover transcription factors binding sites on the DNA from genome-wide surveys like chromatin immunoprecipitation assays on tiling arrays (ChIP-chip). We then use the localization about the binding of TFs to explain expression levels of regulated genes. In this way we identify a regulatory synergy between two TFs, the oncogene C-MYC and SP1. C-MYC and SP1 bind preferentially at promoters and when SP1 binds next to C-NIYC on the DNA, the nearby gene is strongly expressed. The association between the two TFs at promoters is reflected by the binding sites conservation across mammals, by the permissive underlying chromatin states 'it represents an important control mechanism involved in cellular proliferation, thereby involved in cancer. Secondly, we identify the characteristics of TF estrogen receptor alpha (hERa) target genes and we study the influence of hERa in regulating transcription. hERa, upon hormone estrogen signaling, binds to DNA to regulate transcription of its targets in concert with its co-factors. To overcome the scarce experimental data about the binding sites of other TFs that may interact with hERa, we conduct in silico analysis of the sequences underlying the ChIP sites using the collection of position weight matrices (PWMs) of hERa partners, TFs FOXA1 and SP1. We combine ChIP-chip and ChIP-paired-end-diTags (ChIP-pet) data about hERa binding on DNA with the sequence information to explain gene expression levels in a large collection of cancer tissue samples and also on studies about the response of cells to estrogen. We confirm that hERa binding sites are distributed anywhere on the genome. However, we distinguish between binding sites near promoters and binding sites along the transcripts. The first group shows weak binding of hERa and high occurrence of SP1 motifs, in particular near estrogen responsive genes. The second group shows strong binding of hERa and significant correlation between the number of binding sites along a gene and the strength of gene induction in presence of estrogen. Some binding sites of the second group also show presence of FOXA1, but the role of this TF still needs to be investigated. Different mechanisms have been proposed to explain hERa-mediated induction of gene expression. Our work supports the model of hERa activating gene expression from distal binding sites by interacting with promoter bound TFs, like SP1. hERa has been associated with survival rates of breast cancer patients, though explanatory models are still incomplete: this result is important to better understand how hERa can control gene expression. Thirdly, we address the difficult question of regulatory network inference. We tackle this problem analyzing time-series of biological measurements such as quantification of mRNA levels or protein concentrations. Our approach uses the well-established penalized linear regression models where we impose sparseness on the connectivity of the regulatory network. We extend this method enforcing the coherence of the regulatory dependencies: a TF must coherently behave as an activator, or a repressor on all its targets. This requirement is implemented as constraints on the signs of the regressed coefficients in the penalized linear regression model. Our approach is better at reconstructing meaningful biological networks than previous methods based on penalized regression. The method is tested on the DREAM2 challenge of reconstructing a five-genes/TFs regulatory network obtaining the best performance in the "undirected signed excitatory" category. Thus, these bioinformatics methods, which are reliable, interpretable and fast enough to cover large biological dataset, have enabled us to better understand gene regulation in humans.

The hourglass and the early conservation models--co-existing patterns of developmental constraints in vertebrates.

Developmental constraints have been postulated to limit the space of feasible phenotypes and thus shape animal evolution. These constraints have been suggested to be the strongest during either early or mid-embryogenesis, which corresponds to the early conservation model or the hourglass model, respectively. Conflicting results have been reported, but in recent studies of animal transcriptomes the hourglass model has been favored. Studies usually report descriptive statistics calculated for all genes over all developmental time points. This introduces dependencies between the sets of compared genes and may lead to biased results. Here we overcome this problem using an alternative modular analysis. We used the Iterative Signature Algorithm to identify distinct modules of genes co-expressed specifically in consecutive stages of zebrafish development. We then performed a detailed comparison of several gene properties between modules, allowing for a less biased and more powerful analysis. Notably, our analysis corroborated the hourglass pattern at the regulatory level, with sequences of regulatory regions being most conserved for genes expressed in mid-development but not at the level of gene sequence, age, or expression, in contrast to some previous studies. The early conservation model was supported with gene duplication and birth that were the most rare for genes expressed in early development. Finally, for all gene properties, we observed the least conservation for genes expressed in late development or adult, consistent with both models. Overall, with the modular approach, we showed that different levels of molecular evolution follow different patterns of developmental constraints. Thus both models are valid, but with respect to different genomic features.

Classification of urban multi-angular image sequences by aligning their manifolds

When dealing with multi-angular image sequences, problems of reflectance changes due either to illumination and acquisition geometry, or to interactions with the atmosphere, naturally arise. These phenomena interplay with the scene and lead to a modification of the measured radiance: for example, according to the angle of acquisition, tall objects may be seen from top or from the side and different light scatterings may affect the surfaces. This results in shifts in the acquired radiance, that make the problem of multi-angular classification harder and might lead to catastrophic results, since surfaces with the same reflectance return significantly different signals. In this paper, rather than performing atmospheric or bi-directional reflection distribution function (BRDF) correction, a non-linear manifold learning approach is used to align data structures. This method maximizes the similarity between the different acquisitions by deforming their manifold, thus enhancing the transferability of classification models among the images of the sequence.

Hepatic hemangiomas: Factors associated with T2 shine-through effect on diffusion-weighted MR sequences.

PURPOSE: To determine the frequency and factors associated with the presence of T2 shine-through effect in hepatic hemangiomas on diffusion-weighted (DW) magnetic resonance (MR) sequences. MATERIALS AND METHODS: This retrospective study was approved by institutional review board with waiver of informed consent. One hundred forty-nine consecutive patients with 388 hepatic hemangiomas who underwent a liver MR between January 2010 and November 2011 were included. MR analysis evaluated the lesion characteristics (signal intensities and enhancement patterns (classical, rapidly filling, delayed filling)), the presence of T2 shine-through effect on DW sequences (b values of 0, 150, and 600s/mm(2)), and apparent diffusion coefficient (ADC) values. Multivariate analysis was performed to study the factors associated with the T2 shine-through effect. RESULTS: T2 shine-through effect was observed in 204/388 (52.6%) of hepatic hemangiomas and in 100 (67.1%) patients. Mean ADC value of hemangiomas with T2 shine-through effect was significantly lower than hemangiomas without (2.0±0.48 vs 2.38±0.45, P<.0001). On multivariate analysis, high signal intensity on fat-suppressed T2-weighted fast spin-echo images, hemangiomas with classical or delayed enhancement, and the ADC of the liver were the only significant factors associated with T2 shine-through effect. CONCLUSION: T2 shine-through effect is commonly observed in hepatic hemangiomas and is related to hemangiomas characteristics. Radiologists should be aware of this phenomenon which could lead to misdiagnosis. Its presence should not question the diagnosis of hemangiomas when typical MR findings are found.

Differences in glucose transporter gene expression between rat pancreatic alpha- and beta-cells are correlated to differences in glucose transport but not in glucose utilization.

Glucose exerts inverse effects upon the secretory function of islet alpha- and beta-cells, suppressing glucagon release and increasing insulin release. This diverse action may result from differences in glucose transport and metabolism between the two cell types. The present study compares glucose transport in rat alpha- and beta-cells. beta-Cells transcribed GLUT2 and, to a lesser extent, GLUT 1; alpha-cells contained GLUT1 but no GLUT2 mRNA. No other GLUT-like sequences were found among cDNAs from alpha- or beta-cells. Both cell types expressed 43-kDa GLUT1 protein which was enhanced by culture. The 62-kDa beta-cell GLUT2 protein was converted to a 58-kDa protein after trypsin treatment of the cells without detectable consequences upon glucose transport kinetics. In beta-cells, the rates of glucose transport were 10-fold higher than in alpha-cells. In both cell types, glucose uptake exceeded the rates of glucose utilization by a factor of 10 or more. Glycolytic flux, measured as D-[5(3)H]glucose utilization, was comparable in alpha- and beta-cells between 1 and 10 mmol/liter substrate. In conclusion, differences in glucose transporter gene expression between alpha- and beta-cells can be correlated with differences in glucose transport kinetics but not with different glucose utilization rates.

The tumor antigens RHAMM and G250/CAIX are expressed in head and neck squamous cell carcinomas and elicit specific CD8+ T cell responses.

Despite advances in surgery, radio- and chemotherapy, therapeutic approaches for patients with head and neck squamous carcinoma (HNSCC) need to be improved. Immunotherapies eliciting tumor specific immune responses might constitute novel treatment options. We therefore investigated the expression and immunogenicity of two tumor-associated antigens (TAA) the receptor for hyaluronic acid mediated motility (RHAMM) and carboanhydrase IX (G250/CAIX) in HNSCC patients. Twenty-two HNSCC samples were examined for the expression of RHAMM and G250 by Western blotting and immunohistochemistry, 14/22 samples were tested for HLA-A2 expression by flow cytometry. For 8/22 samples single tumor-cell suspensions were generated, and mixed lymphocyte peptide cultures (MLPC) were performed to evaluate the frequencies of cytotoxic T cells specifically recognizing RHAMM and G250 using Tetramer staining/multi-color flow cytometry and enzyme linked immunosorbent spot (ELISPOT) assays. RHAMM and G250 were expressed in 73 and 80% of the HNSCC samples at the protein level. A co-expression of both TAAs could be detected in 60% of the patients. In 4/8 HLA-A2+ patients, 0.06-0.13% of CD8+ effector T cells recognized Tetramers for RHAMM or G250 and secreted IFNgamma and granzyme B in ELISPOT assays. RHAMM and G250 are expressed at high frequency and high protein level in HNSCCs and are recognized by cytotoxic CD8+ effector T cells. Therefore both TAAs constitute interesting targets for T cell based immunotherapies for HNSCC.

Induction of CTL in vivo by major histocompatibility complex class I-peptide complexes covalently associated on the cell surface.

The identification of endogenously produced antigenic peptides presented by MHC class I molecules has opened the way to peptide-based strategies for CTL induction in vivo. Here we demonstrate that the induction in vivo of CTL directed against naturally processed antigens can be triggered by injection of syngeneic cells expressing covalent major histocompatibility complex class I-peptide complexes. In the model system used, the induction of HLA-Cw3 specific cytotoxic T lymphocytes (CTL) in mice by cell surface-associated, covalent H-2Kd (Kd)-Cw3 peptide complexes was investigated. The Kd-restricted Cw3 peptide 170-179 (RYLKNGKETL), which mimics the major natural epitope recognized by Cw3-specific CTL in H-2d mice, was converted to a photoreactive derivative by replacing Arg-170 with N-beta-(4-azidosalicyloyl)-L-2,3-diaminopropionic acid. This peptide derivative was equivalent to the parental Cw3 peptide in terms of binding to Kd molecules and recognition by Cw3-specific CTL clones and could be cross-linked efficiently and selectively to Kd molecules on the surface of Con A-stimulated spleen cells from H-2d mice. Photocross-linking prevented the rapid dissociation of Kd-peptide derivative complexes that takes place under physiological conditions. Cultures of spleen cells or peritoneal exudate cells from mice inoculated i.p. with peptide-pulsed and photocross-linked cells developed a strong CTL response following antigenic stimulation in vitro. The cultured cells efficiently lysed not only target cells sensitized with the Cw3 170-179 peptide but also target cells transfected with the Cw3 gene. Moreover, their TCR preferentially expressed V beta 10 and J alpha pHDS58 segments as well as conserved junctional sequences, as has been observed previously in Cw3-specific CTL responses. In contrast, no Cw3-specific CTL response could be obtained in cultures derived from mice injected with Con A-stimulated spleen cells pulsed with the peptide derivative without photocross-linking.

Cointegrase, a naturally occurring, truncated form of IS21 transposase, catalyzes replicon fusion rather than simple insertion of IS21.

The bacterial insertion sequence IS21 contains two genes, istA and istB, which are organized as an operon. IS21 spontaneously forms tandem repeats designated (IS21)2. Plasmids carrying (IS21)2 react efficiently with other replicons, producing cointegrates via a cut-and-paste mechanism. Here we show that transposition of a single IS21 element (simple insertion) and cointegrate formation involving (IS21)2 result from two distinct non-replicative pathways, which are essentially due to two differentiated IstA proteins, transposase and cointegrase. In Escherichia coli, transposase was characterized as the full-length, 46 kDa product of the istA gene, whereas the 45 kDa cointegrase was expressed, in-frame, from a natural internal translation start of istA. The istB gene, which could be experimentally disconnected from istA, provided a helper protein that strongly stimulated the transposase and cointegrase-driven reactions. Site-directed mutagenesis was used to express either cointegrase or transposase from the istA gene. Cointegrase promoted replicon fusion at high frequencies by acting on IS21 ends which were linked by 2, 3, or 4 bp junction sequences in (IS21)2. By contrast, cointegrase poorly catalyzed simple insertion of IS21 elements. Transposase had intermediate, uniform activity in both pathways. The ability of transposase to synapse two widely spaced IS21 ends may reside in the eight N-terminal amino acid residues which are absent from cointegrase. Given the 2 or 3 bp spacing in naturally occurring IS21 tandems and the specialization of cointegrase, the fulminant spread of IS21 via cointegration can now be understood.

Molecular phylogenetics of soricid shrews (Mammalia) based on mitochondrial cytochrome b gene sequences: with special reference to the Soricinae

Molecular phylogeny of soricid shrews (Soricidae, Eulipotyphla, Mammalia) based on 1140 bp mitochondrial cytochrome b gene (cytb) sequences was inferred by the maximum likelihood (ML) method. All 13 genera of extant Soricinae and two genera of Crocidurinae were included in the analyses. Anourosorex was phylogenetically distant from the main groupings within Soricinae and Crocidurinae in the ML tree. Thus, it could not be determined to which subfamily Anourosorex should be assigned: Soricinae, Crocidurinae or a new subfamily. Soricinae (excluding Anourosorex) should be divided into four tribes: Neomyini, Notiosoricini, Soricini and Blarinini. However, monophyly of Blarinini was not robust in the present data set. Also, branching orders among tribes of Soricinae and those among genera of Neomyini could not be determined because of insufficient phylogenetic information of the cytb sequences. For water shrews of Neomyini (Chimarrogale, Nectogale and Neomys), monophyly of Neomys and the Chimarrogale-Nectogale group could not be verified, which implies the possibility of multiple origins for the semi-aquatic mode of living among taxa within Neomyini. Episoriculus may contain several separate genera. Blarinella was included in Blarinini not Soricini, based on the cytb sequences, but the confidence level was rather low; hence more phylogenetic information is needed to determine its phylogenetic position. Furthermore, some specific problems of taxonomy of soricid shrews were clarified, for example phylogeny of local populations of Notiosorex crawfordi, Chimarrogale himalayica and Crocidura attenuata.

Synergistic action of GA-binding protein and glucocorticoid receptor in transcription from the mouse mammary tumor virus promoter.

B lymphocytes are among the first cells to be infected by mouse mammary tumor virus (MMTV), and they play a crucial role in its life cycle. To study transcriptional regulation of MMTV in B cells, we have analyzed two areas of the long terminal repeat (LTR) next to the glucocorticoid receptor binding site, fp1 (at position -139 to -146 from the cap site) and fp2 (at -157 to -164). Both showed B-cell-specific protection in DNase I in vitro footprinting assays and contain binding sites for Ets transcription factors, a large family of proteins involved in cell proliferation and differentiation and oncogenic transformation. In gel retardation assays, fp1 and fp2 bound the heterodimeric Ets factor GA-binding protein (GABP) present in B-cell nuclear extracts, which was identified by various criteria: formation of dimers and tetramers, sensitivity to pro-oxidant conditions, inhibition of binding by specific antisera, and comigration of complexes with those formed by recombinant GABP. Mutations which prevented complex formation in vitro abolished glucocorticoid-stimulated transcription from an MMTV LTR linked to a reporter gene in transiently transfected B-cell lines, whereas they did not affect the basal level. Exogenously expressed GABP resulted in an increased level of hormone response of the LTR reporter plasmid and produced a synergistic effect with the coexpressed glucocorticoid receptor, indicating cooperation between the two. This is the first example of GABP cooperation with a steroid receptor, providing the opportunity for studying the integration of their intracellular signaling pathways.

Optimization Of Promoter Sequences In Order To Mimic Physiological Pde6b Expression

Purpose: Many retinal degenerations result from defective retina-specific gene expressions. Thus, it is important to understand how the expression of a photoreceptor-specific gene is regulated in vivo in order to achieve successful gene therapy. The present study aims to design an AAV2/8 vector that can regulate the transcript level in a physiological manner to replace missing PDE6b in Rd1 and Rd10 mice. In previous studies (Ogieta, et al., 2000), the short 5' flanking sequence of the human PDE6b gene (350 bp) was shown to be photoreceptor-specific in transgenic mice. However, the efficiency and specificity of the 5' flanking region of the human PDE6b was not investigated in the context of gene therapy during retinal degeneration. In this study, two different sequences of the 5' flanking region of the human PDE6b gene were studied as promoter elements and their expression will be tested in wild type and diseased retinas (Rd 10 mice).Methods: Two 5' flanking fragments of the human PDE6b gene: (-93 to +53 (150 bp) and -297 to +53 (350 bp)) were cloned in different plasmids in order to check their expression in vitro and in vivo by constructing an AAV2/8 vector. These elements drove the activity of either luciferase (pGL3 plasmids) or EGFP. jetPEI transfection in Y 79 cells was used to evaluate gene expression through luciferase activity. Constructs encoding EGFP under the control of the two promoters were performed in AAV2.1-93 (or 297)-EGFP plasmids to produce AAV2/8 vectors.Results: When pGL3-93 (150 bp) or pGL3-297 (350 bp) were transfected in the Y-79 cells, the smaller fragment (150 bp) showed higher gene expression compared to the 350 bp element and to the SV40 control, as previously reported. The 350 bp drove similar levels of expression when compared to the SV40 promoter. In view of these results, the fragments (150 bp or 350 bp) were integrated into the AAV2.1-EGFP plasmid to produce AAV2/8 vector, and we are currently evaluating the efficiency and specificity of the produced constructs in vivo in normal and diseased retinas.Conclusions: Comparisons of these vectors with vectors bearing ubiquitous promoters should reveal which construct is the most suitable to drive efficient and specific gene expression in diseased retinas in order to restore a normal function on the long term.