Semantic Assembly and Annotation of Draft RNAseq Transcripts without a Reference Genome.

Transcriptomes are one of the first sources of high-throughput genomic data that have benefitted from the introduction of Next-Gen Sequencing. As sequencing technology becomes more accessible, transcriptome sequencing is applicable to multiple organisms for which genome sequences are unavailable. Currently all methods for de novo assembly are based on the concept of matching the nucleotide context overlapping between short fragments-reads. However, even short reads may still contain biologically relevant information which can be used as hints in guiding the assembly process. We propose a computational workflow for the reconstruction and functional annotation of expressed gene transcripts that does not require a reference genome sequence and can be tolerant to low coverage, high error rates and other issues that often lead to poor results of de novo assembly in studies of non-model organisms. We start with either raw sequences or the output of a context-based de novo transcriptome assembly. Instead of mapping reads to a reference genome or creating a completely unsupervised clustering of reads, we assemble the unknown transcriptome using nearest homologs from a public database as seeds. We consider even distant relations, indirectly linking protein-coding fragments to entire gene families in multiple distantly related genomes. The intended application of the proposed method is an additional step of semantic (based on relations between protein-coding fragments) scaffolding following traditional (i.e. based on sequence overlap) de novo assembly. The method we developed was effective in analysis of the jellyfish Cyanea capillata transcriptome and may be applicable in other studies of gene expression in species lacking a high quality reference genome sequence. Our algorithms are implemented in C and designed for parallel computation using a high-performance computer. The software is available free of charge via an open source license.

Immunolocalization of a voltage-gated calcium channel ß subunit in the tentacles and cnidocytes of the Portuguese man-of-war, Physalia physalis.

This study investigated the localization of a voltage-gated calcium channel (VGCC) ß subunit in the tentacles and cnidocytes of the Portuguese man-of-war using confocal immunocytochemistry. An antibody specific to the Ca(2+) channel ß subunit of the Portuguese-man-of-war (PpCaVß) was generated, and characterized by Western immunoblotting. The antibody labeling was widespread in the ectoderm of cnidosacs of the tentacles. The binding of the antibody on isolated cnidocytes was distributed at the base of the cell and appeared as multiple strong fluorescent plaques located around the basal hemisphere of the cell. The distribution of PpCaVß in the cnidocyte is consistent with previous studies on other hydrozoans that demonstrated that cnidocytes convey sensory information to other cnidocytes through chemical synapses in which the cnidocyte is pre-synaptic to elements of the animal's nervous system. Importantly and surprisingly, PpCaVß did not localize to the apical surface of the cnidocyte where the exocytotic events involved in cnidocyst discharge are thought to take place.

Physiological and chemical analysis of neurotransmitter candidates at a fast excitatory synapse in the jellyfish Cyanea capillata (Cnidaria, Scyphozoa).

Motor nerve net (MNN) neurons in the jellyfish Cyanea capillata communicate with one another by way of fast, bidirectional excitatory chemical synapses. As is the case with almost all identified chemical synapses in cnidarians, the identity of the neurotransmitter at these synapses is unclear. MNN neurons are large enough for stable intracellular recordings. This, together with the fact that they can be exposed, providing unlimited access to them and to their synapses, prompted a study of the action of a variety of neurotransmitter candidates, including those typically associated with fast synapses in higher animals. Only the amino acids taurine and beta-alanine produced physiological responses consistent with those of the normal EPSP in these cells. Moreover, chemical analysis revealed that both taurine and beta-alanine are present in the neurons and released by depolarization. These various findings strongly suggest that either or both of these amino acids, or a closely related compound is the neurotransmitter at the fast chemical synapses between MNN neurons.

The regulation of cnidocyte discharge.

Because cnidocytes are exceedingly complex cells which can only be used once, their discharge is highly regulated by way of a variety of chemosensory, mechanosensory and endogenous pathways. The integration of these various inputs ultimately results in exocytosis and then discharge of the cnidocyte's diagnostic organelle, the cnidocyst. Here we review what is known about the sensory pathways that regulate cnidocytes, the electrical events that manifest in cnidocytes following sensory stimulation and the ionic mechanisms that underlie discharge.

Chemosensory pathways in the capitate tentacles of the hydroid Cladonema.

The introduction of an extract of Artemia into the sea water bathing tentacles from the hydroid Cladonema triggers a burst of electrical activity that can be recorded intracellularly from cnidocytes in the capitate tentacles. These bursts, which are composed of a variety of events, including action potentials and EPSPs, are Ca2+ dependent, and are abolished by pretreatment with NiCl2, suggesting that voltage-gated Ca2+ channels are involved in their generation or transmission. Intracellular injection of Lucifer Yellow and recordings from pairs of cnidocytes reveal that the cnidocytes are electrically coupled to one another, but that they are not uncoupled by heptanol. The role of these chemosensory pathways in priming the cnidocytes for discharge is discussed.

Evidence for a common pattern of peptidergic innervation of cnidocytes.

Tentacles from representatives of all four classes of the phylum Cnidaria were examined using antibodies against the neuropeptides FMRFamide and RFamide to reveal the organization of neurons and nerve nets associated with cnidocytes. The tentacles of all species examined contained FMRFamide- or RFamide-immunoreactive neurons, in varying densities. In representatives from the Scyphozoa, Hydrozoa, and Cubozoa, the FMRFamide-immunoreactive neurons formed plexuses at the base of the cnidocyte assemblages; in anthozoans, the absence of discrete assemblies of cnidocytes precluded visual co-localization of cnidocytes and immunoreactive neurons. In all four classes, immunoreactive sensory cells connected these peptidergic nerve nets to the surface of the tentacle. These findings suggest that members of all four cnidarian classes share a common organizational pattern, and it is proposed that this peptidergic innervation may be involved in the chemosensory regulation of cnidocyte discharge.

Creation by mutagenesis of a mammalian Ca(2+) channel beta subunit that confers praziquantel sensitivity to a mammalian Ca(2+) channel.

Voltage-gated Ca(2+) channel beta subunits are important modulators of the pore-forming alpha(1) subunit. We have cloned two schistosome beta subunits that confer sensitivity to the antischistosomal drug praziquantel (PZQ) to an otherwise insensitive mammalian alpha(1) subunit. The primary site of beta subunit interaction with alpha(1) subunits is the beta interaction domain (BID). The BID contains two conserved serines (225, 235 in rat beta2a) that constitute consensus sites for protein kinase C phosphorylation. However, these serines are absent in these schistosome beta subunits. Here we show that the capability to confer PZQ sensitivity can be created in the rat beta2a subunit by eliminating both serines in the BID. These results are consistent with, and should help our understanding of, the selective toxicity of PZQ.

Preparation and Properties of Cnidocytes from the Sea Anemone Anthopleura elegantissima.

Cnidocytes were isolated from the tentacles and acrorhagi of Anthopleura elegantissima by enzymatic treatment with papain followed by centrifugation in a Percoll-containing medium to produce a concentrated fraction of these cells. The morphology of the isolated cells, as revealed by light and electron microscopy, showed that these cells had intact plasma membranes and was comparable to that of cells in situ. Comparison of the ability of different substances to induce discharge in isolated and in situ cnidocytes showed that the responsiveness of isolated cells was reduced, but not abolished, compared to in situ cnidocytes. Electrophysiological recordings made from cnidocytes isolated from acrorhagi showed that these cells possess voltage-activated ionic currents, further proof that the isolation procedure did not effect the integrity of the plasma membrane. Discharge did not occur with changes in membrane potential.

The organization and structure of nerve and muscle in the jellyfish Cyanea capillata (coelenterata; scyphozoa).

The perirhopalial tissue and swimming muscle of Cyanea were examined with light microscopical and electron microscopical techniques. The perirhopalial tissue is a thin, triangular septum found on the subumbrellar surface of the animal. It separates part of the gastric canal system from the surrounding seawater, and is bound on two sides by radial muscle bands and on the third, the shorter side, by a rhopalium and the margin of the bell. The ectoderm of the perirhopalial tissue is composed of large, somewhat cuboidal, vacuolated, myoepithelial cells. The muscle tails of these cells form a single layer of radial, smooth muscle. Neurons of the "giant fiber nerve net" (GFNN), which form an extensive net over the perirhopalial tissue, lie at the base of the vacuolated portion of the myoepithelial cells. These neurons are visible in living tissue. The morphology of individual GFNN neurons was examined following intracellular injection of the fluorescent dye Lucifer Yellow. The neurons are usually bipolar and free of branches. At the electron microscope level, one usually finds that the GFNN neurons contain large vacuoles. The other characteristic feature of these cells is that they form symmetrical, or nonpolarized, synapses; that is, synaptic vesicles are found on both sides of the synapse. The swimming muscle is striated and composed of myoepithelial cells. Each myoepithelial cell has several muscle tails, and those of adjacent cells are linked to gether by desmosomes. The endoderm of the perirhopalial tissue also was examined. This investigation of the organization and ultrastructure of the perirhopalial tissue and surrounding muscle was undertaken to provide essential background information for an ongoing physiological study of the GFNN neurons and their synapses.