Gene Discovery and Expression Profiling in the Toxin Producing Marine Diatom, Pseudo-nitzschia multiseries

Katie Rose Boissoneault, Ph.D., 2004
David Housman, Advisor

Toxic algae are a growing concern in the marine environment. The marine diatom, Pseudo-nitzschia multiseries, produces the neurotoxin domoic acid, which is the cause of amnesic shellfish poisoning. The focus of this thesis was the molecular characterization of P. multiseries with the specific goal of identifying genes which may play a significant role in toxin production.
A complementary DNA (cDNA) library and a database of expressed sequence tags (ESTs) were established for P. multiseries. 2552 cDNAs were sequenced, generating a set of 1955 unique contigs, of which 21% demonstrated significant similarity with known protein coding sequences. Among transcripts of interest identified by sequence similarity were fucoxanthin-chlorophyll a/c light harvesting protein, C4-specific pyruvate, orthophosphate dikinase, glutamate dehydrogenase, and 5-oxo-L-prolinase.

Genes whose expression patterns were correlated with toxin production were identified by hybridization to a microarray manufactured from 5376 cDNAs. 121 cDNAs, representing 12 unique cDNA contigs, showed significantly increased expression levels in P. multiseries cell populations actively producing toxin. The up-regulated transcripts included cDNAs with sequence similarity to 3-carboxymuconate cyclase, phosphoenolpyruvate carboxykinase, and an amino acid transporter. Prospects for further application of molecular genetic technology to the understanding of the physiology and ecology of P. multiseries is discussed.