RNA isolation from corals and other cnidarian species using urea-LiCl as a denaturant.

A method of RNA isolation using a solution of urea-LiCl as a denaturing agent was tested on stony coral. As the method does not require homogenization of tissues prior to their incubation in the denaturant, specimen collected in the field can be immediately transferred to the urea-LiCl solution. The method was also tested on tissues of other cnidarian species. RNA was isolated from fresh tissues of jellyfish and sea anemones using two protocols - that is, incubations in the urea-LiCl solution were either performed on homogenized tissues or on intact tissues or specimen. RNA quality was evaluated on a bioanalyser.

A SLC6 transporter cloned from the lion's mane jellyfish (Cnidaria, Scyphozoa) is expressed in neurons.

In the course of recent comparative genomic studies conducted on nervous systems across the phylogeny, current thinking is leaning in favor of more heterogeneity among nervous systems than what was initially expected. The isolation and characterization of molecular components that constitute the cnidarian neuron is not only of interest to the physiologist but also, on a larger scale, to those who study the evolution of nervous systems. Understanding the function of those ancient neurons involves the identification of neurotransmitters and their precursors, the description of nutrients used by neurons for metabolic purposes and the identification of integral membrane proteins that bind to those compounds. Using a molecular cloning strategy targeting membrane proteins that are known to be present in all forms of life, we isolated a member of the solute carrier family 6 from the scyphozoan jellyfish Cyanea capillata. The phylogenetic analysis suggested that the new transporter sequence belongs to an ancestral group of the nutrient amino acid transporter subfamily and is part of a cluster of cnidarian sequences which may translocate the same substrate. We found that the jellyfish transporter is expressed in neurons of the motor nerve net of the animal. To this end, we established an in situ hybridization protocol for the tissues of C. capillata and developed a specific antibody to the jellyfish transporter. Finally, we showed that the gene that codes for the jellyfish transporter also expresses a long non-coding RNA. We hope that this research will contribute to studies that seek to understand what constitutes a neuron in species that belong to an ancient phylum.

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.

Escape variants of the XPR1 gammaretrovirus receptor are rare due to reliance on a splice donor site and a short hypervariable loop.

Entry determinants in the XPR1 receptor for the xenotropic/polytropic mouse leukemia viruses (XP-MLVs) lie in its third and fourth putative extracellular loops (ECLs). The critical ECL3 receptor determinant overlies a splice donor and is evolutionarily conserved in vertebrate XPR1 genes; 2 of the 3 rare replacement mutations at this site destroy this receptor determinant. The 13 residue ECL4 is hypervariable, and replacement mutations carrying an intact ECL3 site alter but do not abolish receptor activity, including replacement of the entire loop with that of a jellyfish (Cnidaria) XPR1. Because ECL4 deletions are found in all X-MLV-infected Mus subspecies, we deleted each ECL4 residue to determine if deletion-associated restriction is residue-specific or is effected by loop size. All deletions influence receptor function, although different deletions affect different XP-MLVs. Thus, receptor usage of a constrained splice site and a loop that tolerates mutations severely limits the likelihood of host escape mutations.

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.

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.

Identification of a novel aminergic-like G protein-coupled receptor in the cnidarian Renilla koellikeri.

Biogenic amines exert various physiological effects in cnidarians, but the receptors involved in these responses are not known. We have cloned a novel G protein-coupled receptor cDNA from an anthozoan, the sea pansy Renilla koellikeri, that shows homology to mammalian catecholamine receptors and, to a lesser extent, to peptidergic receptors. This putative receptor, named Ren2, has a DRC pattern that replaces the well-conserved DRY motif on the cytoplasmic side of the transmembrane III and lacks the cysteine residues usually found in the second extracellular loop and C-terminus tail. Both the second extracellular loop and the N-terminal tail were seen to be short (six and three amino acids, respectively). Northern blot analysis suggests that the receptor gene codes for two transcripts. Localization of these transcripts by in situ hybridization demonstrated abundant expression in the epithelium of the pharyngeal wall, the oral disk and tentacles as well as in the endodermal epithelium lining the gastrovascular cavities.

A new G protein-coupled receptor from a primitive metazoan shows homology with vertebrate aminergic receptors and displays constitutive activity in mammalian cells.

Biogenic amine receptors mediate wide-ranging hormonal and modulatory functions in vertebrates, but are largely unknown in primitive invertebrates. In a representative of the most basal multicellular animals possessing a nervous system, the cnidarian Renilla koellikeri, aminergic-like receptors were previously characterized pharmacologically and found to engender control of the animal's bioluminescent and peristaltic reactions. Using degenerate oligonucleotides in a RT-PCR strategy, we obtained a full-length cDNA encoding a polypeptide with typical G protein-coupled receptor (GPCR) characteristics and which displayed a significant degree of sequence similarity (up to 45%) to biogenic amine receptors, particularly dopamine and adrenergic receptors. The new receptor, named Ren1, did not resemble any one specific type of amine GPCR and thus could not be identified on the basis of sequence. Ren1 was expressed transiently and stably in cultured mammalian cells, as demonstrated by immunocytochemistry and western blotting. Functional analysis of transfected HEK293, LTK- and COS-7 cells, based on both cAMP and Ca2+ signalling assays, revealed that Ren1 was not activated by any of the known biogenic amines tested and several related metabolites. The results indicated, however, that cells stably expressing Ren1 contained, on average, an 11-fold higher level of cAMP than the controls, in the absence of agonist stimulation. The high basal cAMP levels were shown to be specific for Ren1 and to vary proportionally with the level of Ren1 expressed in the transfected cells. Taken together, the data suggested that Ren1 was expressed as a constitutively active receptor. Its identification provides a basis for examination of the early evolutionary emergence of GPCRs and their functional properties.