Marine bioprospecting is the search for new marine bioactive compounds and large-scale screening in extracts represents the traditional approach. Here, we report an alternative complementary protocol, called digital marine bioprospecting, based on deep sequencing of transcriptomes. We sequenced the transcriptomes from the adult polyp stage of two cold-water sea anemones, Bolocera tuediae and Hormathia digitata. We generated approximately 1.1 million quality-filtered sequencing reads by 454 pyrosequencing, which were assembled into approximately 120,000 contigs and 220,000 single reads. Based on annotation and gene ontology analysis we profiled the expressed mRNA transcripts according to known biological processes. As a proof-of-concept we identified polypeptide toxins with a potential blocking activity on sodium and potassium voltage-gated channels from digital transcriptome libraries.
References
[1]
Koehn, F.E.; Carter, G.T. Rediscovering natural products as a source of new drugs. Discov. Med. 2005, 26, 159–164.
[2]
Johansen, S.D.; Emblem, A.; Karlsen, B.O.; Okkenhaug, S.; Hansen, H.; Moum, T.; Coucheron, D.H.; Seternes, O.M. Approaching marine bioprospecting in hexacorals by RNA deep sequencing. N. Biotechnol. 2010, 27, 267–275.
[3]
Yamaguchi, Y.; Hasegawa, Y.; Honma, T.; Nagashima, Y.; Shiomi, K. Screening and cDNA cloning of Kv1 potassium channel toxins in sea anemones. Mar. Drugs 2010, 8, 2893–2905, doi:10.3390/md8122893.
[4]
Sperstad, S.V.; Haug, T.; Blencke, H.M.; Styrvold, O.B.; Li, C.; Stensv？g, K. Antimicrobial peptides from marine invertebrates: Challenges and perspectives in marine antimicrobial peptide discovery. Biotechnol. Adv. 2011, 5, 519–530.
[5]
Vera, J.C.; Wheat, C.W.; Fescemyer, H.W.; Frilander, M.J.; Crawford, D.L.; Hanski, I.; Marden, J.H. Rapid transcriptome characterization for a nonmodel organism using 454 pyrosequencing. Mol. Ecol. 2008, 7, 1636–1647.
[6]
Parchman, T.L.; Geist, K.S.; Grahnen, J.A.; Benkman, C.W.; Buerkle, C.A. Transcriptome sequencing in an ecologically important tree species: Assembly, annotation, and marker discovery. BMC Genomics 2010, 11, 180, doi:10.1186/1471-2164-11-180.
[7]
Wang, Y.; Zeng, X.; Iyer, N.J.; Bryant, D.W.; Mockler, T.C.; Mahalingam, R. Exploring the switchgrass transcriptome using second-generation sequencing technology. PLoS One 2012, 7, 3.
Shinzato, C.; Shoguchi, E.; Kawashima, T.; Hamada, M.; Hisata, K.; Tanaka, M.; Fujie, M.; Fujiwara, M.; Koyanagi, R.; Ikuta, T.; et al. Using the Acropora digitifera genome to understand coral responses to environmental change. Nature 2011, 476, 320–323, doi:10.1038/nature10249.
[11]
Molinski, T.F.; Dalisay, D.S.; Lievens, S.L.; Saludes, J.P. Drug development from marine natural products. Nat. Rev. Drug. Discov. 2009, 8, 69–85, doi:10.1038/nrd2487.
[12]
Rocha, J.; Peixe, L.; Gomes, N.C.M.; Calado, R. Cnidarians as a source of new marine bioactive compounds-an overview of the last decade and future steps for bioprospecting. Mar. Drugs 2011, 9, 1860–1886, doi:10.3390/md9101860.
[13]
Meyer, E.; Aglyamova, G.V.; Wang, S.; Buchanan-Carter, J.; Abrego, D.; Colbourne, J.K.; Willis, B.L.; Matz, M.V. Sequencing and de novo analysis of a coral larval transcriptome using 454 GSFlx. BMC Genomics 2009, 10, 219.
[14]
Polato, N.R.; Vera, J.C.; Baums, I.B. Gene discovery in the threatened elkhorn coral: 454 sequencing of the Acropora palmata transcriptome. PLoS One 2011, 6, 12.
[15]
Traylor-Knowles, N.; Granger, B.R.; Lubinski, T.J.; Parikh, J.R.; Garamszegi, S.; Xia, Y.; Marto, J.A.; Kaufman, L.; Finnerty, J.R. Production of a reference transcriptome and transcriptomic database (PocilloporaBase) for the cauliflower coral, Pocillopora damicornis. BMC Genomics 2011, 12, 585, doi:10.1186/1471-2164-12-585.
[16]
Yuyama, I.; Watanabe, T.; Takei, Y. Profiling differential gene expression of symbiotic and aposymbiotic corals using a high coverage gene expression profiling (HiCEP) analysis. Mar. Biotechnol. 2011, 1, 32–40.
[17]
Weis, V.M.; Davy, S.K.; Hoegh-Guldberg, O.; Rodriguez-Lanetty, M.; Pringle, J.R. Cell biology in model systems as the key to understanding corals. Trends Ecol. Evol. 2008, 7, 369–376.
[18]
Sunagawa, S.; Wilson, E.C.; Thaler, M.; Smith, M.L.; Caruso, C.; Pringle, J.R.; Weis, V.M.; Medina, M.; Schwarz, J.A. Generation and analysis of transcriptomic resources for a model system on the rise: the sea anemone Aiptasia pallida and its dinoflagellate endosymbiont. BMC Genomics 2009, 10, 258, doi:10.1186/1471-2164-10-258.
[19]
Morgan, M.B.; Parker, C.C.; Robinson, J.W.; Pierce, E.M. Using representational difference analysis to detect changes in transcript expression of Aiptasia genes after laboratory exposure to lindane. Aquat. Toxicol. 2012, 110-111, 66–73, doi:10.1016/j.aquatox.2012.01.001.
[20]
Schwarz, J.A.; Brokstein, P.B.; Voolstra, C.; Terry, A.Y.; Manohar, C.F.; Miller, D.J.; Szmant, A.M.; Coffroth, M.A.; Medina, M. Coral life history and symbiosis: Functional genomic resources for two reef building Caribbean corals, Acropora palmata and Montastraea faveolata. BMC Genomics 2008, 9, 97.
[21]
Sabourault, C.; Ganot, P.; Deleury, E.; Allemand, D.; Furla, P. Comprehensive EST analysis of the symbiotic sea anemone, Anemonia viridis. BMC Genomics 2009, 10, 333, doi:10.1186/1471-2164-10-333.
[22]
Norton, R.S. Structure and structure-function relationships of sea anemone proteins that interact with the sodium channel. Toxicon 1991, 29, 1051–1084, doi:10.1016/0041-0101(91)90205-6.
[23]
Handbook of Neurotoxicology; Massaro, E.J., Ed.; Humana Press: Totowa, NJ, USA, 2002; Volume I, p. 685.
[24]
Wunderer, G.; Fritz, H.; Wachter, E.; Machleidt, W. Amino-acid sequence of a coelenterate toxin: Toxin II from Anemonia sulcata. Eur. J. Biochem. 1976, 1, 193–198.
[25]
Anderluh, G.; Podlesek, Z.; Macek, P. A common motif in proparts of Cnidarian toxins and nematocyst collagens and its putative role. Biochim. Biophys. Acta 2000, 1476, 372–376, doi:10.1016/S0167-4838(99)00237-X.
[26]
Stevens, M.; Peigneur, S.; Tytgat, J. Neurotoxins and their binding areas on voltage-gated sodium channels. Front. Pharmacol. 2011, 2, 71.
Lomax, J. Get ready to GO! A biologist’s guide to the gene ontology. Brief. Bioinform. 2005, 6, 298–304, doi:10.1093/bib/6.3.298.
[29]
Emblem, ？. Genomic Analyses of the Cold-Water Coral Lophelia and Sea Anemones. PhD Thesis, University of Troms？, Norway, 2011.
[30]
St Pierre, L.; Fischer, H.; Adams, D.J.; Schenning, M.; Lavidis, N.; de Jersey, J.; Masci, P.P.; Lavin, M.F. Distinct activities of novel neurotoxins from Australian venomous snakes for nicotinic acetylcholine receptors. Cell. Mol. Life Sci. 2007, 21, 2829–2840.
[31]
Kozlov, S.; Malyavka, A.; McCutchen, B.; Lu, A.; Schepers, E.; Herrmann, R.; Grishin, E. A novel strategy for the identification of toxinlike structures in spider venom. Proteins 2005, 59, 131–140, doi:10.1002/prot.20390.
[32]
Kozlov, S.; Grishin, E. The mining of toxin-like polypeptides from EST database by single residue distribution analysis. BMC Genomics 2011, 12, 88, doi:10.1186/1471-2164-12-88.
[33]
Marchler-Bauer, A.; Anderson, J.B.; Chitsaz, F.; Derbyshire, M.K.; DeWeese-Scott, C.; Fong, J.H.; Geer, L.Y.; Geer, R.C.; Gonzales, N.R.; Gwadz, M.; et al. CDD: Specific functional annotation with the Conserved Domain Database. Nucleic Acids Res. 2009, 39, 225–229.
[34]
Wanke, E.; Zaharenko, A.J.; Redaelli, E.; Schiavon, E. Actions of sea anemone type 1 neurotoxins on voltage-gated sodium channel isoforms. Toxicon 2009, 54, 1102–1111, doi:10.1016/j.toxicon.2009.04.018.
Beress, L.; Zwick, J. Purification of two crab-paralyzing polypeptides from the sea anemone Bolocera tuediae. Mar. Chem. 1980, 8, 333–338, doi:10.1016/0304-4203(80)90022-5.
[37]
Dauplais, M.; Lecoq, A.; Song, J.; Cotton, J.; Jamin, N.; Gilquin, B.; Roumestand, C.; Vita, C.; de Medeiros, C.L.; Rowan, E.G. On the convergent evolution of animal toxins. Conservation of a diad of functional residues in potassium channel-blocking toxins with unrelated structures. J. Biol. Chem. 1997, 272, 4302–4309.
[38]
Minagawa, S.; Ishida, M.; Shimakura, K.; Nagashima, Y.; Shiomi, K. Isolation and amino acid sequences of two Kunitz-typeprotease inhibitors from the sea anemone Anthopleura aff. xanthogrammica. Comp. Biochem. Physiol. B Biochem. Mol. Biol. 1997, 118, 381–386, doi:10.1016/S0305-0491(97)00174-0.
Verollet, R. A major step towards efficient sample preparation with bead-beating. Biotechniques 2008, 44, 832–833.
[41]
Conesa, A.; G？tz, S.; Garcia-Gomez, J.M.; Terol, J.; Talon, M.; Robles, M. Blast2GO: A universal tool for annotation, visualization and analysis in functional genomics research. Bioinformatics 2005, 21, 3674–3676, doi:10.1093/bioinformatics/bti610.
[42]
Hu, Z.; Bao, J.; Reecy, J.M. CateGOrizer: A Web-Based Program to Batch Analyze Gene Ontology Classification Categories. Online J. Bioinform. 2008, 9, 108–112.
[43]
CLCbio. Available online: http://www.clcbio.com/ (accessed on 15 August 2012).
[44]
Clustal: Multiple Sequence Alignment. Available online: http://www.clustal.org/ (accessed on 15 August 2012).
[45]
Arnold, K.; Bordoli, L.; Kopp, J.; Schwede, T. The SWISS-MODEL Workspace: A web-based environment for protein structure modelling. Bioinformatics 2006, 22, 195–201, doi:10.1093/bioinformatics/bti770.
[46]
SWISS-MODEL: Swiss Institute of Bioinformatics. Available online: http://swissmodel.expasy.org/ (accessed on 15 August 2012).
[47]
PyMOL: A User-Sponsored Molecular Visualization System on an Open-Source Foundation. Available online: http://www.pymol.org/ (accessed on 15 August 2012).
[48]
POVRAY-Persistence of Vision. Available online: http://www.povray.org/ (accessed on 15 August 2012).
[49]
Bioinformatics Toolkit. Available online: http://toolkit.tuebingen.mpg.de/sixframe (accessed on 15 August 2012).
[50]
Hartley, J.L.; Salehi-Ashtiani, K.; Hill, D.E. Proteome expression moves in vitro: Resources and tools for harnessing the human proteome. Nat. Methods 2008, 5, 1001–1002.
[51]
Goshima, N.; Kawamura, Y.; Fukumoto, A.; Miura, A.; Honma, R.; Satoh, R.; Wakamatsu, A.; Yamamoto, J.; Kimura, K.; Nishikawa, T.; et al. Human protein factory for converting the transcriptome into in vitro-expressed proteome. Nat. Methods 2008, 5, 1011–1017, doi:10.1038/nmeth.1273.
