Polarized blazar X-rays imply particle acceleration in shocks.

Most of the light from blazars, active galactic nuclei with jets of magnetized plasma that point nearly along the line of sight, is produced by high-energy particles, up to around 1 TeV. Although the jets are known to be ultimately powered by a supermassive black hole, how the particles are accelerated to such high energies has been an unanswered question. The process must be related to the magnetic field, which can be probed by observations of the polarization of light from the jets. Measurements of the radio to optical polarization-the only range available until now-probe extended regions of the jet containing particles that left the acceleration site days to years earlier1-3, and hence do not directly explore the acceleration mechanism, as could X-ray measurements. Here we report the detection of X-ray polarization from the blazar Markarian 501 (Mrk 501). We measure an X-ray linear polarization degree ΠX of around 10%, which is a factor of around 2 higher than the value at optical wavelengths, with a polarization angle parallel to the radio jet. This points to a shock front as the source of particle acceleration and also implies that the plasma becomes increasingly turbulent with distance from the shock.

Deeply conserved chordate noncoding sequences preserve genome synteny but do not drive gene duplicate retention.

Animal genomes possess highly conserved cis-regulatory sequences that are often found near genes that regulate transcription and development. Researchers have proposed that the strong conservation of these sequences may affect the evolution of the surrounding genome, both by repressing rearrangement, and possibly by promoting duplicate gene retention. Conflicting data, however, have made the validity of these propositions unclear. Here, we use a new computational method to identify phylogenetically conserved noncoding elements (PCNEs) in a manner that is not biased by rearrangement and duplication. This method is powerful enough to identify more than a thousand PCNEs that have been conserved between vertebrates and the basal chordate amphioxus. We test 42 of our PCNEs in transgenic zebrafish assays--including examples from vertebrates and amphioxus--and find that the majority are functional enhancers. We find that PCNEs are enriched around genes with ancient synteny conservation, and that this association is strongest for extragenic PCNEs, suggesting that cis-regulatory interdigitation plays a key role in repressing genome rearrangement. Next, we classify mouse and zebrafish genes according to association with PCNEs, synteny conservation, duplication history, and presence in bidirectional promoter pairs, and use these data to cluster gene functions into a series of distinct evolutionary patterns. These results demonstrate that subfunctionalization of conserved cis-regulation has not been the primary determinate of gene duplicate retention in vertebrates. Instead, the data support the gene balance hypothesis, which proposes that duplicate retention has been driven by selection against dosage imbalances in genes with many protein connections.

Evolution of a core gene network for skeletogenesis in chordates.

The skeleton is one of the most important features for the reconstruction of vertebrate phylogeny but few data are available to understand its molecular origin. In mammals the Runt genes are central regulators of skeletogenesis. Runx2 was shown to be essential for osteoblast differentiation, tooth development, and bone formation. Both Runx2 and Runx3 are essential for chondrocyte maturation. Furthermore, Runx2 directly regulates Indian hedgehog expression, a master coordinator of skeletal development. To clarify the correlation of Runt gene evolution and the emergence of cartilage and bone in vertebrates, we cloned the Runt genes from hagfish as representative of jawless fish (MgRunxA, MgRunxB) and from dogfish as representative of jawed cartilaginous fish (ScRunx1-3). According to our phylogenetic reconstruction the stem species of chordates harboured a single Runt gene and thereafter Runt locus duplications occurred during early vertebrate evolution. All newly isolated Runt genes were expressed in cartilage according to quantitative PCR. In situ hybridisation confirmed high MgRunxA expression in hard cartilage of hagfish. In dogfish ScRunx2 and ScRunx3 were expressed in embryonal cartilage whereas all three Runt genes were detected in teeth and placoid scales. In cephalochordates (lancelets) Runt, Hedgehog and SoxE were strongly expressed in the gill bars and expression of Runt and Hedgehog was found in endo- as well as ectodermal cells. Furthermore we demonstrate that the lancelet Runt protein binds to Runt binding sites in the lancelet Hedgehog promoter and regulates its activity. Together, these results suggest that Runt and Hedgehog were part of a core gene network for cartilage formation, which was already active in the gill bars of the common ancestor of cephalochordates and vertebrates and diversified after Runt duplications had occurred during vertebrate evolution. The similarities in expression patterns of Runt genes support the view that teeth and placoid scales evolved from a homologous developmental module.

The globin gene family of the cephalochordate amphioxus: implications for chordate globin evolution.

BACKGROUND: The lancelet amphioxus (Cephalochordata) is a close relative of vertebrates and thus may enhance our understanding of vertebrate gene and genome evolution. In this context, the globins are one of the best studied models for gene family evolution. Previous biochemical studies have demonstrated the presence of an intracellular globin in notochord tissue and myotome of amphioxus, but the corresponding gene has not yet been identified. Genomic resources of Branchiostoma floridae now facilitate the identification, experimental confirmation and molecular evolutionary analysis of its globin gene repertoire. RESULTS: We show that B. floridae harbors at least fifteen paralogous globin genes, all of which reveal evidence of gene expression. The protein sequences of twelve globins display the conserved characteristics of a functional globin fold. In phylogenetic analyses, the amphioxus globin BflGb4 forms a common clade with vertebrate neuroglobins, indicating the presence of this nerve globin in cephalochordates. Orthology is corroborated by conserved syntenic linkage of BflGb4 and flanking genes. The kinetics of ligand binding of recombinantly expressed BflGb4 reveals that this globin is hexacoordinated with a high oxygen association rate, thus strongly resembling vertebrate neuroglobin. In addition, possible amphioxus orthologs of the vertebrate globin X lineage and of the myoglobin/cytoglobin/hemoglobin lineage can be identified, including one gene as a candidate for being expressed in notochord tissue. Genomic analyses identify conserved synteny between amphioxus globin-containing regions and the vertebrate ß-globin locus, possibly arguing against a late transpositional origin of the ß-globin cluster in vertebrates. Some amphioxus globin gene structures exhibit minisatellite-like tandem duplications of intron-exon boundaries ("mirages"), which may serve to explain the creation of novel intron positions within the globin genes. CONCLUSIONS: The identification of putative orthologs of vertebrate globin variants in the B. floridae genome underlines the importance of cephalochordates for elucidating vertebrate genome evolution. The present study facilitates detailed functional studies of the amphioxus globins in order to trace conserved properties and specific adaptations of respiratory proteins at the base of chordate evolution.

Timing and mechanism of ancient vertebrate genome duplications -- the adventure of a hypothesis.

Complete genome doubling has long-term consequences for the genome structure and the subsequent evolution of an organism. It has been suggested that two genome duplications occurred at the origin of vertebrates (known as the 2R hypothesis). However, there has been considerable debate as to whether these were two successive duplications, or whether a single duplication occurred, followed by large-scale segmental duplications. In this article, we review and compare the evidence for the 2R duplications from vertebrate genomes with similar data from other more recent polyploids.

Data integration using scanners with SQL output--the bioscanners project at sourceforge.

There is currently no standardized approach for parsing output that the numerous bioinformatical tools generate. Because the framework approach of the Bio-toolkits has some shortcomings, we searched for alternative approaches. To this end, we evaluated scanner generators for various programming languages with respect to their potential of standalone, small and fast applications that can easily delivered on any modern and many ancient operating system. We developed sample applications that generate standard SQL database code and thereby greatly simplify the parsing work of data integration and data analysis. At the sourceforge project page the source code and some binaries for a selection of our applications are freely available at http://bioscanners.sourceforge.net.

Polyploidy and genome restructuring: a variety of outcomes.

Dramatic genome rearrangement has been observed after whole genome duplication (WGD) in some plant species, leading many to suggest that genome restructuring may be a common consequence of WGD. However, recent analyses of ancient WGDs in yeast and vertebrates have not shown any evidence for increased rearrangement after WGD. When WGD events across all three kingdoms of eukaryotic life are considered-including plants, yeast, vertebrates, and human cancers-we find that a variety of outcomes are possible, from genome restructuring to genome stasis. In fact, striking differences in genome change after WGD can be observed within single plant genera, indicating that there are no simple rules that can predict a genome's reaction to WGD.

Monte Carlo analysis of an ODE Model of the Sea Urchin Endomesoderm Network.

BACKGROUND: Gene Regulatory Networks (GRNs) control the differentiation, specification and function of cells at the genomic level. The levels of interactions within large GRNs are of enormous depth and complexity. Details about many GRNs are emerging, but in most cases it is unknown to what extent they control a given process, i.e. the grade of completeness is uncertain. This uncertainty stems from limited experimental data, which is the main bottleneck for creating detailed dynamical models of cellular processes. Parameter estimation for each node is often infeasible for very large GRNs. We propose a method, based on random parameter estimations through Monte-Carlo simulations to measure completeness grades of GRNs. RESULTS: We developed a heuristic to assess the completeness of large GRNs, using ODE simulations under different conditions and randomly sampled parameter sets to detect parameter-invariant effects of perturbations. To test this heuristic, we constructed the first ODE model of the whole sea urchin endomesoderm GRN, one of the best studied large GRNs. We find that nearly 48% of the parameter-invariant effects correspond with experimental data, which is 65% of the expected optimal agreement obtained from a submodel for which kinetic parameters were estimated and used for simulations. Randomized versions of the model reproduce only 23.5% of the experimental data. CONCLUSION: The method described in this paper enables an evaluation of network topologies of GRNs without requiring any parameter values. The benefit of this method is exemplified in the first mathematical analysis of the complete Endomesoderm Network Model. The predictions we provide deliver candidate nodes in the network that are likely to be erroneous or miss unknown connections, which may need additional experiments to improve the network topology. This mathematical model can serve as a scaffold for detailed and more realistic models. We propose that our method can be used to assess a completeness grade of any GRN. This could be especially useful for GRNs involved in human diseases, where often the amount of connectivity is unknown and/or many genes/interactions are missing.

GOblet: annotation of anonymous sequence data with gene ontology and pathway terms.

The functional annotation of genomic data has become a major task for the ever-growing number of sequencing projects. In order to address this challenge, we recently developed GOblet, a free web service for the annotation of anonymous sequences with Gene Ontology (GO) terms. However, to overcome limitations of the GO terminology, and to aid in understanding not only single components but as well systemic interactions between the individual components, we have now extended the GOblet web service to integrate also pathway annotations. Furthermore, we extended and upgraded the data analysis pipeline with improved summaries, and added term enrichment and clustering algorithms. Finally, we are now making GOblet available as a stand-alone application for high-throughput processing on local machines. The advantages of this frequently requested feature is that a) the user can avoid restrictions of our web service for uploading and processing large amounts of data, and that b) confidential data can be analysed without insecure transfer to a public web server. The stand-alone version of the web service has been implemented using platform independent Tcl-scripts, which can be run with just a single runtime file utilizing the Starkit technology. The GOblet web service and the stand-alone application are freely available at http://goblet.molgen.mpg.de.

Early vertebrate whole genome duplications were predated by a period of intense genome rearrangement.

Researchers, supported by data from polyploid plants, have suggested that whole genome duplication (WGD) may induce genomic instability and rearrangement, an idea which could have important implications for vertebrate evolution. Benefiting from the newly released amphioxus genome sequence (Branchiostoma floridae), an invertebrate that researchers have hoped is representative of the ancestral chordate genome, we have used gene proximity conservation to estimate rates of genome rearrangement throughout vertebrates and some of their invertebrate ancestors. We find that, while amphioxus remains the best single source of invertebrate information about the early chordate genome, its genome structure is not particularly well conserved and it cannot be considered a fossilization of the vertebrate preduplication genome. In agreement with previous reports, we identify two WGD events in early vertebrates and another in teleost fish. However, we find that the early vertebrate WGD events were not followed by increased rates of genome rearrangement. Indeed, we measure massive genome rearrangement prior to these WGD events. We propose that the vertebrate WGD events may have been symptoms of a preexisting predisposition toward genomic structural change.

A global view of gene expression in lithium and zinc treated sea urchin embryos: new components of gene regulatory networks.

BACKGROUND: The genome of the sea urchin Strongylocentrotus purpuratus has recently been sequenced because it is a major model system for the study of gene regulatory networks. Embryonic expression patterns for most genes are unknown, however. RESULTS: Using large-scale screens on arrays carrying 50% to 70% of all genes, we identified novel territory-specific markers. Our strategy was based on computational selection of genes that are differentially expressed in lithium-treated embryos, which form excess endomesoderm, and in zinc-treated embryos, in which endomesoderm specification is blocked. Whole-mount in situ hybridization (WISH) analysis of 700 genes indicates that the apical organ region is eliminated in lithium-treated embryos. Conversely, apical and specifically neural markers are expressed more broadly in zinc-treated embryos, whereas endomesoderm signaling is severely reduced. Strikingly, the number of serotonergic neurons is amplified by at least tenfold in zinc-treated embryos. WISH analysis further indicates that there is crosstalk between the Wnt (wingless int), Notch, and fibroblast growth factor signaling pathways in secondary mesoderm cell specification and differentiation, similar to signaling cascades that function during development of presomitic mesoderm in mouse embryogenesis. We provide differential expression data for more than 4,000 genes and WISH patterns of more than 250 genes, and more than 2,400 annotated WISH images. CONCLUSION: Our work provides tissue-specific expression patterns for a large fraction of the sea urchin genes that have not yet been included in existing regulatory networks and await functional integration. Furthermore, we noted neuron-inducing activity of zinc on embryonic development; this is the first observation of such activity in any organism.

The genome of the sea urchin Strongylocentrotus purpuratus.

We report the sequence and analysis of the 814-megabase genome of the sea urchin Strongylocentrotus purpuratus, a model for developmental and systems biology. The sequencing strategy combined whole-genome shotgun and bacterial artificial chromosome (BAC) sequences. This use of BAC clones, aided by a pooling strategy, overcame difficulties associated with high heterozygosity of the genome. The genome encodes about 23,300 genes, including many previously thought to be vertebrate innovations or known only outside the deuterostomes. This echinoderm genome provides an evolutionary outgroup for the chordates and yields insights into the evolution of deuterostomes.

The amphioxus Hairy family: differential fate after duplication.

Vertebrate Hairy genes are highly pleiotropic and have been implicated in numerous functions, such as somitogenesis, neurogenesis and endocrine tissue development. In order to gain insight into the timing of acquisition of these roles by the Hairy subfamily, we have cloned and studied the expression pattern of the Hairy gene(s) in amphioxus. The cephalochordate amphioxus is widely believed to be the living invertebrate more closely related to vertebrates, the genome of which has not undergone the massive gene duplications that took place early during vertebrate evolution. Surprisingly, we have isolated eight Hairy genes from the 'pre-duplicative' amphioxus genome. In situ hybridisation on amphioxus embryos showed that Hairy genes had experienced a process of subfunctionalisation that is predicted in the DDC model (for duplication-degeneration-complementation). Only the summation of four out of the eight Amphi-Hairy genes expression resembles the expression pattern of vertebrate Hairy genes, i.e. in the central nervous system, presomitic mesoderm, somites, notochord and gut. In addition, Amphi-Hairy genes expression suggest that amphioxus early somites are molecularly prefigured in an anteroposterior sequence in the dorsolateral wall of the archenteron, and the presence of a midbrain/hindbrain boundary. The expansion of the amphioxus Hairy subfamily request for caution when deducing the evolutionary history of a gene family in chordates based in the singularity of the amphioxus genome. Amphioxus may resemble the ancestor of the vertebrates, but it is not the ancestor, only its closest living relative, a privileged position that should not assume the freezing of its genome.

On the origin of the chordate central nervous system: expression of onecut in the sea urchin embryo.

We identified a transcription factor of the onecut class in the sea urchin Strongylocentrotus purpuratus that represents an ortholog of the mammalian gene HNF6, the founding member of the onecut class of proteins. The isolated sea urchin gene, named SpOnecut, encodes a protein of 483 amino acids with one cut domain and a homeodomain. Phylogenetic analysis clearly places the sea urchin gene into this family, most closely related to the ascidian onecut gene HNF-6. Nevertheless, phylogenetic analysis reveals a difficult phylogeny indicating that certain members of the family evolve more rapidly than others and also that the cut domain and homeodomain evolve at a different pace. In fly, worm, ascidian, and teleost fish, the onecut genes isolated so far are exclusively expressed in cells of the central nervous system (CNS), whereas in mammals the two copies of the gene have acquired additional functions in liver and pancreas development. In the sea urchin embryo, expression is first detected in the emerging ciliary band at the late blastula stage. During the gastrula stage, expression is limited to the ciliary band. In the early pluteus stage, SpOnecut is expressed at the apical organ and the elongating arms but continues most prominently in the ciliary band. This is the first gene known that exclusively marks the ciliary band and therein the apical organ in a pluteus larva, whereas chordate orthologs execute essential functions in dorsal CNS development. The significance of this finding for the hypothesis that the ciliary bands and apical organs of the hypothetical "dipleurula"-like chordate ancestor and the chordate/vertebrate CNS are of common origin is discussed.

New evidence for genome-wide duplications at the origin of vertebrates using an amphioxus gene set and completed animal genomes.

The 2R hypothesis predicting two genome duplications at the origin of vertebrates is highly controversial. Studies published so far include limited sequence data from organisms close to the hypothesized genome duplications. Through the comparison of a gene catalog from amphioxus, the closest living invertebrate relative of vertebrates, to 3453 single-copy genes orthologous between Caenorhabditis elegans (C), Drosophila melanogaster (D), and Saccharomyces cerevisiae (Y), and to Ciona intestinalis ESTs, mouse, and human genes, we show with a large number of genes that the gene duplication activity is significantly higher after the separation of amphioxus and the vertebrate lineages, which we estimate at 650 million years (Myr). The majority of human orthologs of 195 CDY groups that could be dated by the molecular clock appear to be duplicated between 300 and 680 Myr with a mean at 488 million years ago (Mya). We detected 485 duplicated chromosomal segments in the human genome containing CDY orthologs, 331 of which are found duplicated in the mouse genome and within regions syntenic between human and mouse, indicating that these were generated earlier than the human-mouse split. Model based calculations of the codon substitution rate of the human genes included in these segments agree with the molecular clock duplication time-scale prediction. Our results favor at least one large duplication event at the origin of vertebrates, followed by smaller scale duplication closer to the bird-mammalian split.

Generation, annotation, evolutionary analysis, and database integration of 20,000 unique sea urchin EST clusters.

Together with the hemichordates, sea urchins represent basal groups of nonchordate invertebrate deuterostomes that occupy a key position in bilaterian evolution. Because sea urchin embryos are also amenable to functional studies, the sea urchin system has emerged as one of the leading models for the analysis of the function of genomic regulatory networks that control development. We have analyzed a total of 107,283 cDNA clones of libraries that span the development of the sea urchin Strongylocentrotus purpuratus. Normalization by oligonucleotide fingerprinting, EST sequencing and sequence clustering resulted in an EST catalog comprised of 20,000 unique genes or gene fragments. Around 7000 of the unique EST consensus sequences were associated with molecular and developmental functions. Phylogenetic comparison of the identified genes to the genome of the urochordate Ciona intestinalis indicate that at least one quarter of the genes thought to be chordate specific were already present at the base of deuterostome evolution. Comparison of the number of gene copies in sea urchins to those in chordates and vertebrates indicates that the sea urchin genome has not undergone extensive gene or complete genome duplications. The established unique gene set represents an essential tool for the annotation and assembly of the forthcoming sea urchin genome sequence. All cDNA clones and filters of all analyzed libraries are available from the resource center of the German genome project at http://www.rzpd.de.