Gene Expression in Florida Red Tide Dinoflagellate Karenia brevis: Analysis of an Expressed Sequence Tag Library and Development of DNA Microarray Kristy B. Lidie,1,2 James C. Ryan,1 Michele Barbier,1* Frances M. Van Dolah1,2 1Marine Biotoxins Program, NOAA Center for Coastal Environmental Health and Biomolecular Research, 219 Fort Johnson Rd., Charleston, SC 29412, U.S.A. 2Marine Biomedicine and Environmental Sciences, Medical University of South Carolina, Charleston, South Carolina, U.S.A. Statione Biologique, Roscoff, France Received: 27 September 2004 / Accepted: 15 December 2004 / Online publication: 15 June 2005 Abstract the microarray has been validated for probe speci- ficity and reproducibility. This is the first informa- Karenia brevis (Davis) is the dinoflagellate respon- tion to be developed on the expressed genome of K. sible for nearly annual red tides in the Gulf of brevis and provides the basis from which to begin Mexico. Although the mechanisms regulating the functional genomic studies on this harmful algal growth and toxicity of this problematic organism are bloom species. of considerable interest, little information is avail- able on its molecular biology. We therefore con- Key words: Karenia brevis — dinoflagellate — ex- structed a complementary DNA library from which pressed sequence tag — microarray — Florida red to gain insight into its expressed genome and to tide — functional genomics develop tools for studying its gene expression. Large- scale sequencing yielded 7001 high-quality expressed sequence tags (ESTs), which clustered into 5280 unique gene groups. The vast majority of genes expressed fell into a low-abundance class, with the highest expressed gene accounting for only 1% of the Introduction total ESTs. Approximately 29% of genes were found to have similarity to known sequences in other Dinoflagellates are unicellular protists most closely organisms after BLAST similarity comparisons to related to the ciliates and apicomplexa (Fast et al., the GenBank public protein database using a cutoff 2002). Unique among eukaryotes, dinoflagellates P )4 of < 10e . We identified for the first time in a have permanently condensed chromatin, but lack dinoflagellate a suite of conserved eukaryotic genes histones and nucleosomes typically involved in involved in cell cycle control, intracellular signaling, regulating chromosome condensation and gene and the transcription and translation machinery. At expression. They also have evolved a unique mitosis least 40 of gene clusters displayed single nucleo- % in which the nuclear envelope remains intact and tide polymorphisms, suggesting the presence of the mitotic spindle consists of extra nuclear micro- multiple gene copies. The average GC content of tubules that traverse the nucleus through cytoplas- ESTs was 51%, with a slight preference for G or C in mic channels (Bhaud et al., 1999). Dinoflagellates the third codon position (53.5%). The ESTs were typically possess large genomes (up to 200 pg/cell) used to develop an oligonucleotide microarray con- that are generally considered to be haploid (Triplett taining 4629 unique features and 3462 replicate et al., 1993; Santos and Coffroth, 2003). probes. Microarray labeling has been optimized, and Karenia brevis is a dinoflagellate whose ex- pressed genome is of interest because of its role in producing the harmful algal blooms (HABs) or ‘‘red tides’’ that occur annually in the Gulf of Mexico. K. *Present address: Statione Biologique, Roscoff, France Correspondence to: Frances M. Van Dolah; E-mail: fran.vandolah@ brevis blooms cause extensive fish kills, mortality noaa.gov among of protected marine mammals, and human DOI: 10.1007/s10126-004-4110-6 Volume 7, 481–493 (2005) Ó Springer Science+Business Media, Inc. 2005 481 482 KRISTY B. LIDIE ET AL.: GENE EXPRESSION IN KARENIA BREVIS illness through the production of highly potent nition of exon and intron boundaries, delineation of neurotoxins known as brevetoxins. The K. brevis protein families, and development of probes for ge- genome consists of 121 chromosomes (Walker, 1982) nomewide expression profiling. To this end we containing 100 pg of DNA per cell, or approximately constructed a cDNA library to K. brevis and carried 1 · 1011 bp (Kim and Martin, 1974; Rizzo, 1982; Si- out large-scale sequencing to yield an expressed EST gee, 1986; Kamykowski et al., 1998). This is 30 times database containing 7001 ESTs and 5280 unique the size of the human genome; however, dinofla- gene clusters. These ESTs were then used to develop gellate chromosomes consist of a permanently con- an oligonucleotide microarray specific for K. brevis densed, genetically inactive central region with gene expression. Microarray technology provides the peripheral loops of B-DNA that protrude from this capacity to profile genomewide changes in gene core and comprise the actively transcribed DNA expression in response to different exposure condi- (Sigee, 1984; Anderson et al., 1992; Bhaud et al., tions, to identify genes involved in specific pathways 1999). Therefore, although the size of the expressed on the basis of their coordinated responses, and to genome of K. brevis is unknown, it is anticipated to assign function to unknown genes on the basis of be substantially smaller than its total genome size their induction in response to known challenges. might predict. This approach will greatly expand our understanding Insight into the molecular mechanisms that of K. brevis physiology at the molecular level and control growth, toxicity, and persistence of K. brevis develop our understanding of the effects of modifi- blooms is critical to understanding the formation of cations on the marine environment (Jenny et al., HABs; however, few investigations into the molec- 2002). Here we present details of cDNA library ular biology of K. brevis exist. Antibody-based ap- construction, insight into the K. brevis genome as proaches have yielded some insight into K. brevis revealed by EST data analysis, and the development cell cycle control, with the identification of the and validation of a DNA microarray for investigation central eukaryotic cell cycle regulator, cyclin- of K. brevis functional genomics. dependent kinase (Van Dolah and Leighfield, 1999), and its regulatory subunit cyclin (Barbier et al., Materials and Methods 2003). Nonetheless the partners and cell cycle sub- strates of this central regulator remain unidentified, Strain and Culture Conditions of Cells. The Wilson and the unique features of the dinoflagellate nucleus isolate of K. brevis was used for this study. The suggest that unusual mechanisms may have growth and behavior of this isolate have been well evolved. The basis for K. brevis toxicity is the pro- studied during the approximately 50 years it has duction of brevetoxins, polyether toxins with struc- been in culture. Cells were maintained in batch tures that suggest synthesis through a polyketide culture in 1-L glass bottles with autoclaved, 20-lm- synthase pathway. Yet investigations into polyke- filtered seawater at 36 psu obtained from a seawater tide synthase genes in K. brevis have resulted in system at the Florida Institute of Technology field ambiguity regarding the dinoflagellate-versus-bacte- station at Vero Beach. Seawater was enriched with f/ rial contributions to polyketide synthase activity in 2 medium (Guillard, 1973). Cultures were main- this organism (Snyder et al., 2003). Antibody-based tained at 25° ±1°C on a 16:8-hour light-dark cycle. studies have also been used to identify stress pro- Illumination from cool white lights was maintained teins that may play a role in the adaptation and at a photon flux density of 40 to 50 lEÆm)2Æs)1 persistence of blooms under stressful environmental (measured by Li-Cor 2p sensor). conditions (Miller-Morey and Van Dolah, 2004). However, the scope of such studies is limited by the Construction of cDNA Library. Cultures were availability of antibodies cross-reactive with dino- harvested by centrifugation (1000g) during the log- flagellate proteins. Thus the need is clear for geno- arithmic phase of growth at circadian time CT 16– mic tools with which to study gene expression and 18. This time was chosen to maximize the likeli- regulation in this organism for which little molec- hood of expression of cell cycle genes, according to ular information is available. the known diel phasing of the K. brevis cell cycle Sequencing of complementary DNA libraries to (Van Dolah and Leighfield, 1999). Total RNA (2 mg) generate expressed sequence tags (ESTs) is an effec- was isolated from approximately 20 L of K. brevis tive means of discovering expressed genes in organ- culture using Qiagen RNeasy columns. Then isms for which genomic data are unavailable. ESTs cDNAs were synthesized by oligo(dT) priming from serve as markers for genes expressed under specific poly(A) messenger RNA, size-selected (>400 bp), conditions and can be used as probes in the recovery and directionally cloned into a k Zap II vector sys- of full-length cDNA or genomic sequences, recog- tem (Stratagene). KRISTY B. LIDIE ET AL.: GENE EXPRESSION IN KARENIA BREVIS 483 Clone Propagation, Plasmid Isolation, and EST contigs to 4629, to which 60-mer oligonucleotides Sequencing. Packaged phages were used to infect were designed. The resulting oligonucleotide probes XL1-Blue cells, and mass in vivo excision of the and controls were printed on glass slides using an pBluescript SK()) phagemid from the k ZAP II vector Ink Jet–based printing method, yielding an 8455 was performed with