OLI Grant: Identification of Differentially Regulated Genes in the Red Tide Dinoflagellate Alexandrium fundyense using Massively Parellel Signature Sequencing (MPSS)

Donald M. Anderson and Deanna L. Erdner,
Biology

Grant Funded: 2002

Proposed Research

"Blooms" of toxic dinoflagellates from several genera result in outbreaks of paralytic shellfish poisoning (PSP), one of the more serious of the global phenomena commonly called red tides or harmful algal blooms. The economic, public health, and ecosystem impacts of PSP outbreaks take a variety of forms, and include human intoxications and death from contaminated shellfish or fish, alterations of marine trophic structure, and death of marine mammals, fish, and seabirds. These impacts are caused by the saxitoxins, a family of neurotoxins produced by some dinoflagellates that accumulate in zooplankton, shellfish, or fish during feeding.

While the chemical structure and activity of saxitoxins (STX) have been characterized, little is known about their biosynthetic pathway or metabolic role within the dinoflagellate. To better understand the regulation of toxin production in dinoflagellates, our laboratory has attempted to isolate gene(s) involved in toxin synthesis or its regulation in Alexandrium fundyense, a toxic dinoflagellate responsible for PSP outbreaks in the northeastern U.S. This has been a challenging task, however, as the use of conventional genetic tools is limited by biological and technical factors associated with dinoflagellates. These include a lack of information on the number of copies of ?toxin genes? and the extraordinarily high content of DNA per cell. Our efforts to identify STX genes have focused on the identification and cloning of differentially expressed gene transcripts, utilizing methods that detect the presence or absence of a gene under a particular condition. These methods have been unsuccessful due to DNA sequence differences between our toxic strains and the closely related non-toxic strains to which we compared them.

To circumvent this problem, we propose to employ a recently developed method to identify genes whose expression is increased or decreased (not just present or absent) under particular conditions. Massively Parallel Signature Sequencing (MPSS) generates a short (17 nucleotide), diagnostic sequence ?tag? for one million individual gene transcripts in a cell, allowing the identification, comparison and quantification of the full cellular complement of expressed genes. In order to maximize the data obtained from the MPSS analysis, we will use cells grown under nitrogen and phosphorus starvation, conditions that decrease and increase cellular toxin content respectively. We then should be able to identify genes for toxin production, N stress and P stress, as sequences specific to each of these conditions should be differentially regulated between the two samples. MPSS analysis is extremely powerful, and will likely be used by several laboratories at WHOI once the procedures are demonstrated on a model system such as ours. Simply stated, no other approach currently available will give us the number of expressed sequence tags for a lower cost or in a more practical format. MPSS is costly, however, and funds from the Ocean Life Institute are needed if we are to take advantage of this extremely powerful new technology to address several longstanding issues in harmful algal bloom ecology. Knowledge of toxin-associated genes will help to clarify the thus far elusive STX biosynthetic pathway in dinoflagellates and facilitate studies on the regulation of STX production, thereby greatly augmenting our understanding of toxic bloom formation. In addition, the STX gene sequences will enable the design of molecular probes that unequivocally identify only toxic cells in a complex natural assemblage of plankton, and these are of use in academic field research as well as in commercial and governmental shellfish toxicity monitoring programs.

Progress Report

Funding from the Ocean Life Institute has enabled us to take advantage of an extremely powerful new technology to address a longstanding issue in harmful algal bloom ecology: the identification of the genes for biosynthesis of the dinoflagellate neurotoxin saxitoxin. Production of this toxin by dinoflagellates from several genera result in outbreaks of paralytic shellfish poisoning (PSP), one of the more serious of the global phenomena commonly called red tides or harmful algal blooms. The economic, public health, and ecosystem impacts of PSP outbreaks take a variety of forms, and include human intoxications and death from contaminated shellfish or fish, alterations of marine trophic structure, and death of marine mammals, fish, and seabirds. Identification of toxin-associated genes will help to clarify the thus far elusive STX biosynthetic pathway in dinoflagellates and facilitate studies on the regulation of STX production, thereby greatly augmenting our understanding of toxic bloom formation. In addition, the STX gene sequences will enable the design of molecular probes that unequivocally identify only toxic cells in a complex natural assemblage of plankton, and these are of use in academic field research as well as in commercial and governmental shellfish toxicity monitoring programs.

Our experimental approach employed a recently developed method to identify genes whose expression is increased or decreased under particular conditions. Massively Parallel Signature Sequencing (MPSS) generates a short (17 nucleotide), diagnostic sequence ?tag? for one million individual gene transcripts in a cell, allowing the identification, comparison and quantification of the full cellular complement of expressed genes. In order to maximize the data obtained from the OLI-funded MPSS analysis, we used cells grown under nitrogen and phosphorus starvation, conditions that decrease and increase cellular toxin content respectively. The results should allow us to identify genes for toxin production, N stress and P stress, as sequences specific to each of these conditions should be differentially regulated between the two samples.

Preliminary analysis of the data provides some intriguing glimpses into dinoflagellate gene regulation. Alexandrium cells contain many more signature tags (~40,000) than do humans (~25,000) or Arabadopsis (~16,000-17,000). Expression levels of a large number of these signatures are significantly different between N- and P-limited conditions (3172 tags at p<0.001). Many signatures are unique to one condition or the other (at p<0.001); 526 signatures are found only under -P conditions and 1412 are unique to N starvation. Data for all signatures that are differentially expressed at p<0.001 are displayed graphically below. Efforts are currently underway to obtain additional sequence information via 3? RACE and EST sequencing of cDNA libraries for genes that are unique to or more highly expressed under P starvation. This includes 1265 signatures at p<0.001 and is expected to include genes associated with toxin synthesis and the P starvation response.