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A Comprehensive Comparative Genomic Approach for Identifying the Saxitoxin Synthesis Genes

This study will use the latest molecular and computational methods to identify and characterize the genes involved in saxitoxin production, the causative agent of Paralytic Shellfish Poisoning (PSP).

Anabaena circinalis The specific aims represent a novel approach for identifying these genes and are only possible with the advent of new technologies that allow rapid and inexpensive DNA sequencing. We propose to obtain 20X genome coverage of three strains of toxic cyanobacteria and near complete transcriptome profiling of three toxic dinoflagellate strains. The cyanobacteria represent an attractive model for identifying putative saxitoxin genes due to their small genomes and the wealth of genomic information available in the public databases.

In contrast, the most significant impacts from saxitoxin toxicity derive from dinoflagellate blooms in marine systems. For these reasons, our project is formulated to use both phylogenomic and molecular data to first identify putative saxitoxin genes in cyanobacteria and then utilize this information to identify homologs in dinoflagellates.

Furthermore, the addition of six new genome−level data sets to public databases will provide unique opportunities for our group as well as other scientists to examine genome evolution and organization, important biochemical pathways, and the mechanisms and extent of horizontal gene transfer (HGT) between microbes. The wealth of sequence information obtained in this study will be combined with data from a recently funded proposal to sequence the transcriptomes of two other dinoflagellates, allowing, for the first time, detailed insights into dinoflagellate evolution and the number and diversity of dinoflagellate genes.

Specific Aims

  1. Using 454 Life Sciences™ GS FLX sequencing technology, complete to high coverage (~20X) the genome sequence of the STX−producing cyanobacterium, Anabaena circinalis, and obtain a similar level of coverage for the genomes of two other toxic cyanobacteria, Cylindrospermopsis raciborskii, and Aphanizomenon issatschenkoi.

  2. Using 454 Life Sciences™ GS FLX technologies, significantly extend our preliminary Expressed sequence tag (EST) data set from Alexandrium tamarense to assemble a transcriptome database for this dinoflagellate and two under−studied saxitoxin−producing dinoflagellates, Pyrodinium bahamense var compressum and Alexandrium minutum.

  3. Identify unique saxitoxin genes in cyanobacteria and dinoflagellates using computational methods and phylogenomic methods and perform pathway analysis to confirm putative saxitoxin genes.

    • Using comparative phylogenomic analysis we will identify a candidate set of saxitoxin genes. Pathway analysis will narrow the set of target genes to those involving substrates used in saxitoxin synthesis (e.g., arginine, acetate, and S−adenosylmethionine−CH3) as well those that carry outspecific tailoring reactions (e.g., N−hydroxylation, sulfation).

  4. Utilize computational methods and phylogenomic methods to develop a better understanding of genome organization, genome evolution, and the mechanisms of the Horizontal gene transfer (HGT) between microbes.

Last modified $Date: 2009/06/28 01:08:46 $
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