Project description:Previously, transcriptomics data for mollusc has been obtained by whole-brain bulk RNA-seq and low-throughput scRNA-seq. We want to construct the first molluscan high-throughput single-neuron transcriptomes for Berghia stephanieae. Around 129,000 cells were collected from 20 brains and the libraries were constructed using the 10X Genomics' Chromium platform. The brains were separated into two samples: the main brain (i.e., the cerebropleural, pedal, buccal ganglion) and the rhinophore ganglion sample. After library preparation, around 1,000 cells were receovered and sequenced. After data analysis, the cells formed eight clusters with marker genes for each cluster identified. Various cell populations that express a wide range of both small-molecule neurotransmitters and neuropeptides such as serotonergic, small cardioactive peptide (SCP), APGWamide, and FMRFamide cells were also identified in the dataset. Interestingly, cells from the rhinophore ganglion of Berghia exhibit great cell heterogeneity, with cells splitting into two general categories and four distinct clusters. The project produced a single-cell dissociation protocol that can be adapted for use in other nudibranch molluscs and a custom data analysis pipeline for data of this nature.
Project description:The abundant and widespread coccolithophore Emiliania huxleyi plays an important role in mediating CO2 exchange between the ocean and the atmosphere through its impact on marine photosynthesis and calcification. Here, we use long serial analysis of gene expression (SAGE) to identify E. huxleyi genes responsive to nitrogen (N) or phosphorus (P) starvation. Long SAGE is an elegant approach for examining quantitative and comprehensive gene expression patterns without a priori knowledge of gene sequences via the detection of 21-bp nucleotide sequence tags. E. huxleyi appears to have a robust transcriptional-level response to macronutrient deficiency, with 42 tags uniquely present or up-regulated twofold or greater in the N-starved library and 128 tags uniquely present or up-regulated twofold or greater in the P-starved library. The expression patterns of several tags were validated with reverse transcriptase PCR. Roughly 48% of these differentially expressed tags could be mapped to publicly available genomic or expressed sequence tag (EST) sequence data. For example, in the P-starved library a number of the tags mapped to genes with a role in P scavenging, including a putative phosphate-repressible permease and a putative polyphosphate synthetase. In short, the long SAGE analyses have (i) identified many new differentially regulated gene sequences, (ii) assigned regulation data to EST sequences with no database homology and unknown function, and (iii) highlighted previously uncharacterized aspects of E. huxleyi N and P physiology. To this end, our long SAGE libraries provide a new public resource for gene discovery and transcriptional analysis in this biogeochemically important marine organism. Keywords: Emiliania, gene expression, nutrients, SAGE, phosphate, nitrogen
Project description:The abundant and widespread coccolithophore Emiliania huxleyi plays an important role in mediating CO2 exchange between the ocean and the atmosphere through its impact on marine photosynthesis and calcification. Here, we use long serial analysis of gene expression (SAGE) to identify E. huxleyi genes responsive to nitrogen (N) or phosphorus (P) starvation. Long SAGE is an elegant approach for examining quantitative and comprehensive gene expression patterns without a priori knowledge of gene sequences via the detection of 21-bp nucleotide sequence tags. E. huxleyi appears to have a robust transcriptional-level response to macronutrient deficiency, with 42 tags uniquely present or up-regulated twofold or greater in the N-starved library and 128 tags uniquely present or up-regulated twofold or greater in the P-starved library. The expression patterns of several tags were validated with reverse transcriptase PCR. Roughly 48% of these differentially expressed tags could be mapped to publicly available genomic or expressed sequence tag (EST) sequence data. For example, in the P-starved library a number of the tags mapped to genes with a role in P scavenging, including a putative phosphate-repressible permease and a putative polyphosphate synthetase. In short, the long SAGE analyses have (i) identified many new differentially regulated gene sequences, (ii) assigned regulation data to EST sequences with no database homology and unknown function, and (iii) highlighted previously uncharacterized aspects of E. huxleyi N and P physiology. To this end, our long SAGE libraries provide a new public resource for gene discovery and transcriptional analysis in this biogeochemically important marine organism. Keywords: Emiliania, gene expression, nutrients, SAGE, phosphate, nitrogen Emiliania huxleyi CCMP 1516 was obtained from the Provasoli-Guillard Center for the Culture of Marine Phytoplankton, Bigelow Laboratories. Cultures were grown at 18°C on a 14 h:10 h light:dark cycle (140 µmol quanta m-2 s-1). Nitrogen and phosphate replete (Replete: 35 µM NO3- and 1.5 µM PO43-), low nitrogen (-N: 10 µM NO3-) and low phosphate (-P: 0 µM PO43-) cells were grown in f/50 medium without Si. Locally collected seawater was filtered (pore size, 0.2 µm) and autoclaved. Filter-sterilized inorganic nutrients, trace metals and vitamins (thiamin, biotin and B12) were added after autoclaving. The cells were grown in 8 L batch cultures. The growth of cultures was monitored daily by cell number counted on a hemacytometer, and by relative fluorescence using a Turner Designs AU Fluorometer. Replete cells were harvested in mid-log phase while –N and –P cells were harvested at the onset of stationary phase for SAGE analysis.
Project description:A novel protein, soritesidine (SOR) with potent toxicity was isolated from the marine sponge Spongosorites sp. SOR exhibited wide range of toxicities over various organisms and cells including brine shrimp (Artemia salina) larvae, sea hare (Aplysia kurodai) eggs, mice, and cultured mammalian cells. Toxicities of SOR were extraordinary potent. It killed mice at 5 ng/mouse after intracerebroventricular (i.c.v.) injection, and brine shrimp and at 0.34 µg/mL. Cytotoxicity for cultured mammalian cancer cell lines against HeLa and L1210 cells were determined to be 0.062 and 12.11 ng/mL, respectively. The SOR-containing fraction cleaved plasmid DNA in a metal ion dependent manner showing genotoxicity of SOR. Purified SOR exhibited molecular weight of 108.7 kDa in MALDI-TOF MS data and isoelectric point of approximately 4.5. N-terminal amino acid sequence up to the 25th residue was determined by Edman degradation. Internal amino acid sequences for fifteen peptides isolated from the enzyme digest of SOR were also determined. None of those amino acid sequences showed similarity to existing proteins, suggesting that SOR is a new proteinous toxin.