Characterization of Allelopathic Compounds Produced by Akashiwo sanguinea.

LaTasha Amisial^1,2, Peter Moeller ^1,3, and Alan Lewitus ^1,2,4

¹ Marine Biomedicine and Environmental Sciences Center, Medical University of South Carolina, Charleston, SC

² Hollings Marine Laboratory, Charleston, SC, USA

³ Toxin Chemistry, National Ocean Services (NOS), Charleston, SC, USA

⁴ Baruch Institute, University of South Carolina, and Marine Resources Research Institute, Charleston, SC, USA

Akashiwo sanguinea (formerly Gymnodinium sanguineum, Gynodinium nelsonii, Gymnodinium splendens) is a dinoflagellate common to coastal marine and estuarine waters where it forms blooms. Although these blooms have been associated with harmful effects to fish and shellfish, the mechanism for toxicity has scantly been examined up tell now and as such is still unknown. Evidence that A. sanguinea produces allelopathic compounds was obtained during a 1980 bloom off southern California, from which macrozooplankton avoidance and reduced grazing were observed. However, chemical characterization and isolation of these allelopathic compounds was not attempted. This thesis research seeks to identify the allelopathic compounds produced by a local South Carolina strain of A. sanguinea, and characterize the mechanism for toxicity. Based on toxins associated with other free-living phototrophic dinoflagellates, the allelopathic compounds are most likely to be protein phosphatases, carbohydrate derivatives, polyethers, and/or polyunsaturated fatty acids. The identification of these compounds will proceed by extracting and isolating the compounds from the A. sanguinea cultures. Every aqueous and organic extract fraction will be tested for toxic activity using a MTT cell viability assay and a standard marine toxicity assay. Once the active fractions have been identified, further purification will be done via HPLC, prep TLC, and column chromatography. The purified compound(s) will then be chemically characterized using NMR, Mass Spectrometry, and elemental analysis. Along with chemical characterization, the LC₅₀ value of the active compound(s) will be determined using a standard marine toxin assay (e.g. abalone larvae mortality). Lastly the species will be tested for the polyketide synthase (PKS) gene, commonly found in other toxic dinoflagellates. Our goal is to establish the mechanism and parameters of toxicity for A. sanguinea. This will assist in the over all understanding of how this common dinoflagellate impacts on ecosystems and natural resources around the world.

Circadian Control of the Cell Cycle in the Dinoflagellate Kareniaa brevis: A Role for Blue Light and Characteristics of a Blue Light Receptor

Stephanie A. Brunelle^1,2,3 and Frances M. Van Dolah^1,2

¹ Marine Biomedicine and Environmental Sciences Center, Medical University of South Carolina, Charleston, SC

² Marine Biotoxins Program, NOAA, Center for Coastal Environmental Health and Biomolecular Research, Charleston, ³ Graduate Program in Marine Biology, College of Charleston, Charleston, SC

The molecular mechanisms controlling the cell cycle in the Florida red tide dinoflagellate, Karenia brevis, are of interest because they ultimately regulate the rate of formation of toxic algal blooms. Previous work in our laboratory has shown that the cell cycle in K. brevis is phased to the diel cycle, such that cells enter the cell cycle at precise times relative to the onset of light. Here, we demonstrate that the cell cycle is under control of a circadian rhythm that is entrained by the dark/light transition. In a number of organisms, blue light serves to entrain circadian rhythms. Therefore, we next investigated the effect of red and blue light on cell cycle progression. In the presence of blue light, K.brevis appears to enter S-phase early, whereas in red light, cell cycle progression is delayed in S-phase entry. This suggests the presence of both blue and red light signaling pathways in K. brevis. Here we characterize a blue-light receptor identified through EST (expressed sequence tag) screening of a K. brevis cDNA library. Cryptochromes are blue-light receptors found in bacteria, plants and animals. The K. brevis ESTs have highest homology to a newly identified class of cryptochrome called Cry DASH. Phylogenetic analysis of the photolyase/blue light receptor gene family shows that the K .brevis cryptochrome falls within the cryptochrome DASH clade and not the photolyase, cryptochrome 1 or cryptochrome 2 clades. Members of the Cry DASH class are generally localized to the mitochondria or chloroplast and have DNA binding activity suggestive of a transcriptional regulatory activity. Other classes of cryptochromes have been shown to be under circadian control, with rhythmic oscillations in gene expression. K. brevis Cry DASH did not display either diel or circadian changes in transcription as assessed using quantitative Real-Time PCR. This is the first blue light receptor to be identified in a dinoflagellate. Extensive high throughput sequencing (25,000 sequences) of two K. brevis cDNA libraries, one prepared from cells harvested during the dark-phase and one from the light-phase of the diel cycle, has failed to identify a sequence with homology to known red light receptors.

Establishment of Epidermal Cell Lines Derived from the Skin of the Atlantic Bottlenose Dolphin (Tursiops truncatus).

Blake C. Ellis¹, Jin Yu¹, Mark S. Kindy^1,2,6, John E. Baatz^1,2,3, Margie Peden-Adams^1,3, Tara J. Ellingham⁴, Daynna J. Wolff⁴, Patricia A. Fair^1,2,7, and Sebastiano Gattoni-Celli^1,5,6*

¹ Marine Biomedicine and Environmental Sciences Center, Medical University of South Carolina, Charleston, SC

² Department of Neurosciences and Neuroscience Institute, Medical University of South Carolina, Charleston, SC

³ Department of Pediatrics, Medical University of South Carolina, Charleston, SC

⁴ Department of Pathology and Laboratory Medicine, Medical University of South Carolina, Charleston, SC

⁵ Department of Radiation Oncology, Medical University of South Carolina, Charleston, SC

⁶ Ralph H. Johnson VA Medical Center, Charleston, SC

⁷ NOAA/National Ocean Service, Center for Coastal Environmental Health and Biomolecular Research, Charleston, SC

The Atlantic bottlenose dolphin (BND) has become a focus of attention as an indicator of the environmental conditions of Atlantic coastal waters because of pathologies and diseases previously unseen in these marine mammals. Humans are exposed to these same waters suggesting that dolphins may be sentinels for human health as well, especially considering similarities in life span, offspring number, and growth and maturity rates between the two species. Since the epidermis serves as the critical interface between the dolphin and its aquatic environment, we have established BND epidermal cell cultures and cell lines from skin tissue as an in vitro tool for evaluating environmental stressors on this protected marine mammal. We have characterized the cell cultures by karyotype analysis, immunohistochemical staining for cytokeratin, and two-dimensional polyacrylamide gel electrophoresis, all of which revealed similar patterns between cell cultures and skin tissue. We also observed that the cytokeratin staining pattern was analogous to that of the human keratinocyte cell line, HaCaT. On the other hand, dolphin cells demonstrated a greater tolerance to high salinity conditions compared to HaCaT cells as indicated by morphology assessment and MTT assay; this is not entirely surprising considering the natural environments of humans and dolphins. Cell lines were obtained by transfection of the dolphin skin cell cultures with a plasmid encoding the SV40 small t and large T antigens in conjunction with the neomyocin-resistance gene. Neomyocin-resistant clones exhibited a marked increase in growth rate compared to the non-transfected cell cultures. The availability of BND epidermal cell cultures and cell lines provides a much needed tool for comparing the responses of marine organisms and humans to environmental stressors as well as a novel experimental approach to studying the dolphin.

Health Effects of Perfluorinated Contaminants in Otariids

Jocelyn R. Flanary¹, Margie M. Peden-Adams^1,2, Thomas Walle^1,3, John R. Kucklick^1,4, and Paul R. Becker, ^1,4

¹ Marine Biomedicine and Environmental Sciences Center, Medical University of South Carolina, Charleston, SC

² Department of Pediatrics, Medical University of South Carolina, Charleston, SC

³ Department of Pharmacology, Medical University of South Carolina, Charleston, SC

⁴ National Institute of Standards and Technology, Hollings Marine Laboratory, Charleston, SC

In recent years a new class of emerging contaminants, perfluorinated compounds (PFCs), have come under investigation. In contrast to most classes of organic contaminants (e.g. polychlorinated biphenyls and pesticides), PFCs are hydrophilic and circulate in the blood and liver upon exposure. Previous studies have shown that PFCs behave as peroxisome proliferators and rodents exposed to them develop cancer. PFCs have worldwide distribution leading to marine mammal exposure; however, at this time very little information is known regarding the health of marine mammals, especially in regard to these contaminants. The health of otariid species are of concern because they include endangered and vulnerable, as well as stable populations. Also, contaminated animals may pose a risk to human exposure, as some otariids are hunted for human consumption. We propose that perfluorinated compounds bind to proteins in the blood and liver of otariids causing protein modifications and disruption of normal cellular function. To test the hypothesis three experimental aims will be performed. First, identification of proteins that bind to perfluorinated compounds, the binding affinities, and resultant protein modifications will be evaluated. The identification of binding proteins will allow for an evaluation of off-loading and PFC half-life as measured from nails, whiskers, scat, and milk samples. Second, an assessment of PFC burdens in blood and liver samples will be obtained as baseline data in evaluating the level of exposure in otariid populations. Third, cellular response to PFCs will be evaluated with dose-response and other toxicity tests, blood chemistry, and histology. In otariids, these data will determine the effects of perfluorinated compounds, the level of risk these compounds pose to otariid populations, and the risk to humans consuming them. This research will also build on the existing knowledge of marine mammal health.

The Role of Leptin in Pulmonary Surfactant Production Using a Seal Model

 
Christina L. Grek1, John E. Baatz1,2, Ailsa J. Hall3, and John A. Hammond4

¹ Marine Biomedicine and Environmental Sciences Center, Medical University of South Carolina, Charleston, SC, USA

² Department of Pediatrics, Medical University of South Carolina, Charleston, SC, USA

³ Sea Mammal Research Unit, Gatty Marine Laboratory, University of St Andrews, St Andrews, Scotland

⁴ Stanford University, Departments of Structural Biology, Microbiology and Immunology, Stanford University School of Medicine, Stanford, CA USA

Leptin, the product of the ob gene, is a circulating cytokine that has been characterized to be secreted primarily by adipocytes in correlation with body mass index and the control of energy balance. Recently leptin and the leptin receptor have been identified in fetal and adult pulmonary tissues. Evidence has been presented that leptin expression is of key importance in the stretch-induced signaling pathway that results in pulmonary surfactant production as well as in lung growth and maturation and in the direct stimulation of surfactant phospholipids. However, the leptin signaling pathway appears to be switched off after full lung development. Surfactant has a variety of physiological and immune functions that includes reducing alveolar surface tension and bacterial clearance. In diving marine mammals surfactant production is key to lung reinflation after collapse during long dives. After diving, seals have been shown to produce amplified surfactant amounts which can be further correlated to high pulmonary leptin protein expression. This suggests that adult seals retain the ability to secrete pulmonary leptin and further proposes the use of the seal lung as a model for human surfactant regulation. We hypothesize that pulmonary leptin expression directly modulates the production and expression of surfactant and is a critical component involved in maintaining human pulmonary health during birth as proposed by a seal model. Using in vivo and in vitro studies leptin, leptin receptor, and surfactant expression and production will be systematically characterized and evaluated by simulating various stressors including oxygen deprivation, carbon dioxide derived acidosis, steroid modulation and pressure variation. The ultimate results from these experiments will provide for insight into the source, triggering mechanisms and pathways by which leptin and associated surfactant expression is occurring.

Structure, Function, and Evolution of E-Protein Transcription Factors Driving Transcription of the Immunoglobulin Heavy Chain Locus Enhancer in Teleost Fish

Jun-ichi Hikima¹, Mara L. Lennard¹, Melanie R. Wilson², Norman W. Miller², L. William Clem², and Gregory W. Warr¹

¹ Marine Biomedicine and Environmental Sciences Center, Medical University of South Carolina, Charleston, SC

and Hollings Marine Laboratory, Charleston, SC

² University of Mississippi Medical Center, Jackson, MS

E-proteins of the class I basic helix-loop-helix (bHLH) family (E2A, HEB, E2-2), are of wide tissue distribution in mammals, typically exert their effects by binding to mE5 sites, and are involved in the development of the immune system. We have investigated the evolution of transcriptional control in the immunoglobulin heavy chain (IgH) locus of vertebrates, using as a model the transcriptional enhancer, Em3’, of the IgH locus of the channel catfish, Ictalurus punctatus. This enhancer functions through the interaction of transcription factors binding to multiple octamer motifs and to a single consensus mE5 site. Two catfish E-protein homologs, related to E2A and HEB were identified as highly expressed in a catfish B cell cDNA library. Regions homologous to the bHLH and activation domains of other vertebrate E-proteins were readily identified in these sequences. The catfish E-protein messages are ubiquitously expressed, being readily detected in catfish B cells, T cells, kidney, spleen, brain, and muscle. The catfish E-proteins strongly activated transcription of a mE5-dependent construct in catfish B cells, and also stimulated transcriptional activation from the core region of the catfish Em3’ enhancer. All E-protein homologues were capable of binding the mE5 motif, and of forming both homo- and heterodimers with other E-proteins. The ability of catfish E2A to homodimerize distinguishes it from its most closely-related mammalian homolog, E12. Comparative analysis of E-protein genes between Fugu and human revealed evolutionary differences. Two E2A genes were identified in Fugu; one of these showed the alternative processing of RNA transcripts and was identified as the ortholog of the human E2A (E12/E47) gene products, whereas the second Fugu E2A gene had no inferred alternative splice products. Overall, our results indicate that, although E-proteins have been highly conserved in vertebrate evolution, a detailed examination of structure/function relationships (as exemplified in the case of E2A) reveals significant evolutionary divergence within the vertebrates. Supported by awards R01 GM62317 and R01 AI19530 from the NIH.

Isolation and Determination of Structure and Biological Activity of a Toxin Produced by the Dinoflagellate, Alexandrium monilatum

Michelle H. Hsia^1,3, Steve Morton^1,2, and Peter D.R. Moeller^1,3

¹ Marine Biomedicine and Environmental Sciences Center, Medical University of South Carolina, Charleston, SC

² Marine Biotoxins, National Ocean Service, Charleston, SC

³ Toxin Chemistry, National Ocean Service, Charleston, SC

The chain-forming dinoflagellate Alexandrium monilatum has been reported to be associated with widespread discolored water and increased fish mortality in the Mississippi Sound and off the eastern and western coasts of Florida. Previous studies over the last 70 years have determined that A. monilatum produces a harmful substance(s) that is predominantly contained in the cell mass as exhibited by apparent increased toxicity when the organism cytolyses. The current research in our lab corroborated earlier research demonstrating that A. monilatum produces a lipophilic toxin, unlike its Alexandrium relatives noted for their production of saxitoxin-like toxins. Using sophisticated chemical, chromatographic, and analytical techniques, we have successfully purified and identified the molecular structure of the toxin produced by A. monilatum. We utilized a 500MHz NMR to carry out a number of experiments (i.e. ¹H, ¹³C, COSY, HSQC, HMBC) to unambiguously determine the molecular structure of the toxin. In addition, we performed mass analysis of the toxin using ESI-MS, MALDI-TOF MS, and Q-TOF mass spectral techniques. The toxin is representative of a polyether macrolide with an empirical formula of C₄₃H₆₀O₁₂. This toxic compound is shown to be identical to a toxin identified as goniodomin A, which is produced by a Japanese tide-pool Alexandrium species.

Function and Regulation of the Cyanotoxin, Microcystin

Wesley C. Jackson, Jr.^1,2 and Alan J. Lewitus^1,2,3

¹ Marine Biomedicine and Environmental Sciences Center, Medical University of South Carolina, Charleston, SC

² Hollings Marine Laboratory, Charleston, SC

³ University of South Carolina and South Carolina Department of Natural Resources, Charleston, SC

Microcystins are potent human liver toxins and carcinogens produced by cyanobacteria in freshwater to brackish environments. The biochemical pathway that produces microcystins has been determined, but little is known about the regulation of this non-ribosomal pathway. Active transport of microcystin out of the cyanobacterial cell does not appear to occur, suggesting an unknown internal function for this compound. The highest concentrations of microcystin are found in the thylakoid, implying a role in photosynthesis. This project tests two hypotheses related to the possible function of microcystin in photosynthesis: Hypothesis 1) microcystin participates in the regulation of photosynthesis through interaction with blue light receptor molecules in a melatonin-like circadian fashion; Hypothesis 2) microcystin plays a role in dissipation of excess energy produced through the photosynthetic process.

To test these hypotheses, differences in genetic regulation and protein composition of two Microcystis aeruginosa cultures are being compared: a microcystin producer, UTEX 2385, and one that reportedly does not produce microcystin, UTEX 2386. Microcystin concentrations will be determined in methanol extracts of these cultures, using High Performance Liquid Chromatography (HPLC) and Enzyme-Linked Immunosorbent Assay (ELISA). To determine genetic differences in these Microcystis cultures, DNA will be extracted using the DNeasy extraction technique. Real-time PCR will be used to sequence both cultures for genetic differences. Finally, known autophosphorylating proteins regulated by full spectrum and blue light regulation (eg. phototropins) are to be determined using comparative Matrix Assisted Laser Desorption Ionization Mass Spectrometry (MALDI-MS). The results from this research will advance understanding of the physiological function of microcystin and the environmental conditions that promote its production. This information is important to predicting cyanobacterial bloom toxicity, and could aid in bloom mitigation.

Oyster Metallothioneins: Unprecedented Diversity in a Metazoan Species

Matthew J. Jenny¹, Gregory W. Warr^1,2, Amy H. Ringwood³, Kevin Schey⁴, and Robert W. Chapman^1,5

¹ Marine Biomedicine and Environmental Sciences Center, Medical University of South Carolina, Charleston, SC

² Department of Biochemistry and Molecular Biology, Medical University of South Carolina, Charleston, SC

³ Department of Biology, University of North Carolina, Charlotte, NC

⁴ Department of Cell and Molecular Pharmacology & Experimental Therapeutics, Medical University of South Carolina, Charleston, SC

⁵ Marine Resources Research Institute, South Carolina Department of Natural Resources, Charleston, SC

Metallothioneins (MTs) are low molecular weight metal-binding proteins typically comprised of two domains (a & b) with characteristic repeating cysteine motifs (Cys-Xn-Cys). They play primary roles in metal metabolism, homeostasis and detoxification. The two domains of MTs are capable of binding metals independently of each other and are functionally diverse. The a-domain is believed to convey structure and stability to the protein while the b-domain participates in metal exchange reactions with zinc and copper-requiring apoproteins. Utilizing molecular genetic and protein biochemical approaches, we have identified three MT gene families in the American oyster, Crassostrea virginica, with distinct differences in primary structure, domain organization, and patterns of expression, including their inducibility by metal ions and other stressors. The first family of oyster MTs (CvMT-I & II) consists of the traditional ab-domain structure, as well as a sub-family of MTs, which appears to have resulted from a mutation followed by a series of exon duplications and consists only of one to four a-domains. We have also described a new molluscan MT family (CvMT-III) characterized by the presence of two b-domains and the absence of a-domains. A third MT gene family (CvMT-IV) was isolated from hemocytes by subtractive hybridization techniques and appears to be more closely related to the classic ab-domain MTs. However, a series of amino acid substitutions has resulted in the occurrence of four additional cysteines which form novel motifs. Southern blot analysis suggests the presence of at least 6 CvMT-I & II genes, and 2-3 copies of both CvMT-III and CvMT-IV. Characterization of a 10X BAC library has identified a single locus for the CvMT-I, II & IV gene families and different loci for CvMT-III. The diversity of oyster MTs appears to have resulted from the evolution of two distinct ancestral MT genes, an unprecedented observation from any metazoan taxon.

Microbial Community Analysis of a Gorgonian Coral, Pseudopterogorgia americana

Christopher Johnston¹, Garriet W. Smith², Cheryl M. Woodley^1,3, and Pamela J. Morris^1,3

¹ Marine Biomedicine and Environmental Sciences Center, Medical University of South Carolina, Charleston, SC

² Department of Biology, University of South Carolina Aiken, Aiken, SC

³ Center for Coastal Environmental Health and Biomolecular Research and Hollings Marine Laboratory, Charleston, SC

Coral reefs have long been recognized as the most diverse marine ecosystem; however, relatively little attention has been focused on the prokaryotic diversity associated with coral reefs until recently. Corals harbor an active microbial community in their surface mucopolysaccharide layer (SML) that may be specific to the host coral over spatial and temporal scales. We used both culture-dependent and culture-independent approaches to characterize bacteria associated with the SML of a healthy gorgonian coral, Pseudopterogorgia americana, from Rocky Point Reef, San Salvador, Bahamas. Microorganisms were isolated from the coral SML and microbial community DNA was directly extracted from the SML for 16S rRNA gene analysis. Of 53 isolates obtained, 30 were distinct ribotypes based on 16S rDNA sequence analysis of the V6-V9 regions. Seven of the 30 isolates were Gram-positive, and all of the isolates clustered within the Bacillus, γ- and α-Proteobacteria. We compared four different DNA extraction and purification methods for recovering microbial community DNA directly from SML samples, and determined the efficiency of each method based on total DNA yield and percent recovery from spiked samples. From the microbial community DNA extracts, we amplified the bacterial 16S rRNA genes and examined the community structure through denaturing gradient gel electrophoresis (DGGE) and 16S rDNA cloning. DGGE profiles resulted in 15 major bands, 12 of which were excised, sequenced, and phylogenetically characterized. Of the DGGE sequences obtained, the majority clustered within the β-Proteobacteria. The 16S rDNA clone library was dominated by the genus Sphingomonas, with almost half of the sequences identified belonging to this genus. Thus, we observed little congruency between the bacterial 16S rRNA genes identified using culture-dependent and culture-independent approaches. Additionally, coral SML and SML isolates were screened for antifungal activity in dual culture bioassays and the presence of quorum sensing signaling molecules using two acyl homoserine lactone autoinducer bioassays, thin layer chromatography, and gas chromatography-mass spectrometry. Of the 30 isolates tested, 2 tested positive for antifungal activity against Aspergillus sydowii and 10 isolates tested positive for signal molecules using the autoinducer bioassays. Quorum sensing may provide one mechanism by which coral-associated microorganisms control populations within their communities, and prevent invasions by opportunistic pathogens.

Oct Transcription Factors and their Coactivators: Are They Evolutionarily Conserved?

Mara L. Lennard¹, Jun-ichi Hikima^1,2, Norman W. Miller³, Melanie R. Wilson³, L. William Clem³, and Gregory W. Warr^1,2

¹ Marine Biomedicine and Environmental Sciences Center, Medical University of South Carolina, Charleston, SC

² Department of Biochemistry and Molecular Biology, Medical University of South Carolina, Charleston, SC

³ Department of Microbiology, University of Mississippi Medical Center, Jackson, MS

A homologue to the POU domain family protein Oct1 has been cloned from a catfish macrophage library. Phylogenetic analysis indicates that the nucleotide sequence is most closely related to that of zebrafish Oct1, and that the catfish molecule is clearly a homologue of mammalian Oct1. Analysis of the inferred protein sequence indicates a high degree of similarity between catfish Oct1 and both zebrafish and mouse Oct1, ranging from 45% in the putative activation domains to as high as 96% in the DNA-binding POU domain. Surprisingly, upon co-transfection (with an octamer-dependent reporter construct) into both mammalian and catfish B cell lines, catfish Oct1 failed to drive transcription. This lack of activity appears to be independent of cell type or reporter construct. Co-transfection of catfish Oct1 with mammalian Bob-1, a known co-activator, yielded similar results. Subsequent transfection studies revealed that catfish Oct1 is acting as a negative regulator of transcription, consistent with the fact that it binds the cognate octamer motif, as measured by electrophoretic mobility shift assays. Thus, it is concluded that the catfish Oct1 homologue is acting differently than its mammalian counterparts, in that it is transcriptionally inactive on the classical Pol II promoters we have tested. Supported by award #GM62317 from the National Institutes of Health.

Gene Expression in the Florida Red Tide Dinoflagellate Karenia brevis: Analysis of an Expressed Sequence Tag Library and Development of a DNA Microarray

Kristy B. Lidie^1,2, James C. Ryan², and Frances M. Van Dolah^1,2

¹ Marine Biomedicine and Environmental Sciences Center, Medical University of South Carolina, Charleston, SC

² Marine Biotoxins Program, NOAA Center for Coastal Environmental Health and Biomolecular Research, Charleston, SC

Karenia brevis is a dinoflagellate responsible for toxic red tides in the Gulf of Mexico. Although the mechanisms regulating its growth and toxicity are of considerable interest, little information is available on its molecular biology. We therefore constructed a cDNA library to gain insight into its expressed genome and to develop tools for gene expression studies. Large scale sequencing yielded 7,001 ESTs representing 5,280 unigene clusters. The vast majority of ESTs fall into a low-abundance class, with the highest expressed gene accounting for only 1% of all ESTs. Approximately 29% of genes have similarity to known sequences in GenBank after BLASTx comparisons, p-value cut-off of 10e^-4. We identify for the first time in a dinoflagellate a suite of conserved genes involved in cell cycle control, intracellular signaling and the transcription and translation machinery. At least 40% of gene clusters displayed single nucleotide polymorphisms, suggesting the presence of multiple copies. The ESTs were used to design a 60-mer oligonucleotide microarray. Microarray labeling has been optimized and the microarray has been validated for probe specificity and reproducibility. This is the first information on the expressed genome of a harmful algal species and provides the basis from which to begin functional genomic studies.

Development of a Dolphin cDNA Microarray

Annalaura Mancia¹, Mats Lundqvist¹, Tracy Romano², Greg Bossart³, Patricia Fair⁴, and Gregory W. Warr¹

¹ Marine Biomedicine and Environmental Sciences Center, Medical University of South Carolina, Charleston, SC

² Mystic Aquarium and Institute for Exploration, Mystic, CT

³ Harbor Branch Oceanographic Institution, Inc., Fort Pierce, FL.

⁴ CCEHBR, National Ocean Service, Charleston, SC

The Atlantic Bottlenose Dolphin (Tursiops truncatus) has been proposed as a sentinel species for the health of the marine environment. Dolphins, as top predators, are sensitive to the biointensification effects of marine toxins, pollutants and infectious disease agents. The aim of this project is to generate molecular tools to assess the health of wild dolphins, thereby indicating the status of the local marine environment and providing information for marine resources management. Random Expressed Sequence Tag (EST) clones have been isolated and sequenced from dolphin Peripheral Blood Leukocyte (PBL) cDNA libraries. Two cDNA libraries from known health status dolphin PBL have been generated including an IL-2 and an LPS-stimulated cDNA library, respectively biased towards T and B-cell gene expression. From the two cDNA-libraries 24,000 ESTs were collected and a total of 2200 unigenes have been sequenced and annotated (www.marinegenomics.org). Moreover, genes known to be important in the innate and adaptive immune responses of terrestrial mammals and in responses to stress and contaminant exposure have been targeted for cloning using PCR-based techniques. A total of 62 dolphin genes of known stress or immune function have been cloned by targeted PCR. The 2200 unigenes from the EST collection and the 62 immune-function targeted genes, together with other genes randomly selected without sequencing from the cDNA libraries, have been amplified and used to construct a cDNA microarray representing 3700 dolphin genes in duplicate for a total of 7400. The first set of microarray data were obtained using captive dolphin PBL RNA as probe. Results show the reproducibilit