Gene expression in the dinoflagellate Karenia brevis : Analysis of an expressed sequence tag (EST) library and development of an oligonucleotide microarray

Karenia brevis (Davis) is a unicellular dinoflagellate responsible for nearly annual “red tides” in the Gulf of Mexico. Insight into the molecular mechanisms that control growth, toxicity, and adaptive mechanisms in K. brevis is critical to understanding the formation and persistence of these toxic blooms. However, little information is available on the molecular biology K. brevis . Therefore, we constructed a cDNA library from which to gain insight into its expressed genome and to develop tools for studying gene expression. Large‐scale sequencing of 9728 clones yielded 7001 high‐quality expressed sequence tags (ESTs) for further analysis. The highest expressed gene accounts for only 1% of the total ESTs. Approximately 29% of ESTs were found to have homology to known sequences in other organisms after BLASTx similarity comparisons to the Genbank nonredundant database ( p <10 −4 ). Using a minimum identity of 95% within a 50‐bp region of overlap, the 7001 ESTs were assembled into 5054 contigs. Of the 5054 contigs, 4399 contained only a single sequence. Of those containing >2 ESTs, approximately 40% displayed single nucleotide polymorphisms, suggesting the presence of multiple gene copies in this haploid organism. Gene‐specific 60‐mer oligonucleotides were then designed for each of the 5054 contigs using a Parcel clustering package, version 2.2.8 (Agilent). The resulting oligonucleotides and controls were printed on glass slides to yield an 8000 feature array using inkjet printing technology (Agilent). The 8000 features included 5054 unique features and 2946 secondary probes. Validation experiments to assess system noise, reproducibility, log ratio accuracy, and differential gene expression are underway using Agilent Feature Extraction tools and Rosetta Luminator Gene Expression Analysis software.