Cell cycle dynamics and the physiology of saxitoxin biosynthesis in Alexandrium fundyense (Dinophyceae)
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The mechanism of saxitoxin (STX) biosynthesis in marine dinoflagellates of the genus Alexandrium is still unknown. The aim of this thesis was to analyze novel aspects of toxigenesis during the cell cycle in Alexandrium and to apply molecular techniques to gain new insights on the genetics and regulation of STX biosynthesis. Synchronized cultures of A. fundyense were studied to determine the dynamics of toxin production throughout the cell cycle. Toxin production was discontinuous, was induced by light and always occurred during a period of approximately eight to ten hours in early G1. Analysis of the cell cycle dynamics suggests the existence of two transition points: one at the beginning of G1, which is light-dependent and holds the cells in a Go-like period, and a second one at the end of G1, which is size-dependent and arrests the cells in G1. A model of the cell cycle of A. fundyense is proposed in which progression through the cell cycle can be arrested at two different transition points located in G1 and toxin production is induced by light during G1. The effects of temperature and phosphate limitation on the linkage between changes in the duration of the cell cycle stages and toxicity were studied in semi-continuous cultures of A. fundyense. A direct correlation between G1 duration and toxin content was observed, along with a clear uncoupling of toxin accumulation from the Sand G2 phases of the cell cycle. In both experiments, toxin production rates remained constant for the respective range of conditions, implying that the variations in toxin content observed were a result of increasing periods of biosynthetic activity. Phosphate limitation enhanced toxin production rates and affected interconversions among STX derivatives in several ways: oxidations to yield the hydroxy-series of STXs were phosphate-dependent while sulfatation reactions were not. Differential Display (DD) analysis was applied to the identification of genes that were up- or downregulated during toxigenesis in synchronized cultures of A. fundyense. Three genes were isolated: S-adenosy lhomocysteine hydrolase, methionine aminopeptidase and a histone-like protein. None could be directly correlated to toxigenesis but instead relate to general cellular metabolism.
Submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy at the Massachusetts Institute of Technology and the Woods Hole Oceanographic Institution March 1998
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