Abstract:
Toxic algae are a growing concern in the marine environment. One unique
marine diatom, Pseudo-nitzschia multiseries (Hasle) Hasle, produces the the neurotoxin
domoic acid, which is the cause of amnesic shellfish poisoning. The molecular
characterization of this organism has been limited to date. Therefore, the focus of this
thesis was to identify and initiate characterization of actively expressed genes that control
cell growth and physiology in P. multiseries, with the specific goal of identifying genes
that may play a significant role in toxin production.
The first step in gene discovery was to establish a complementary DNA (cDNA)
library and a database of expressed sequence tags (ESTs) 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 the
genes identified by sequence similarity were several involved in photosynthetic
pathways, including fucoxanthin-chlorophyll a/c light harvesting protein and a
C4-specific pyruvate, orthophosphate dikinase. Several genes that may be involved in
domoic acid synthesis were also revealed through sequence similarity, for example,
glutamate dehydrogenase and 5-oxo-L-prolinase. In addition, the identification of
sequences that appear novel to Pseudo-nitzschia may provide insight into unique aspects
of Pseudo-nitzschia biology, such as toxin production.
Genes whose expression patterns were correlated with toxin production were
identified by hybridization to a micro array manufactured from 5376 cDNAs.
121 cDNAs, representing 12 unique cDNA contigs or non-redundant cDNAs, showed
significantly increased expression levels in P. multiseries cell populations that were
actively producing toxin. The up-regulated transcripts included cDNAs with sequence
similarity to 3-carboxymuconate cyclase, phosphoenolpyruvate carboxykinase, an amino
acid transporter, a small heat shock protein, a long-chain fatty acid Co-A ligase, and an
aldo/keto reductase. These results provide a framework for investigating the control of
toxin production in P. multiseries. These transcripts may also be useful in ecological
field studies in which they may serve as signatures of toxin production. Prospects for
further application of molecular genetic technology to the understanding of the
physiology and ecology of P. multiseries is discussed.
