Regulation of Toxicity in the Dinoflagellate Alexandrium

摘要亚历山大属有毒鞭毛藻的大量繁殖导致麻痹性贝类中毒（PSP）的爆发，这是一种更严重的全球海洋现象，统称为“赤潮”。PSP暴发对经济、公共卫生和生态系统的影响有多种形式，包括人类因食用受污染的贝类或鱼类而中毒和死亡，渔业物种的阶段，以及海洋哺乳动物、鱼类和海鸟的死亡。这些影响是由石笋毒素引起的，石笋毒素是由鞭毛藻产生的一种神经毒素，在浮游动物、贝类或鱼类的摄食过程中积累。该项目的总体目标是确定亚历山大菌和其他产生石蜡毒素的生物中涉及毒素产生的基因，并研究它们被调节的方式。该方法将使用差异显示来识别细胞之间产生毒素能力不同的信使RNA差异。差异表达的序列可以用来筛选推定的毒素基因在大范围的有毒和无毒物种的鞭毛藻和蓝藻。已鉴定基因毒素特异性的其他间接证据将来自使用抑制剂在不同水平上阻断毒素生物合成的鞭毛藻代谢的操纵。如果由于毒素基因在转录水平上不受调控而导致差异显示方法不成功，则将传统的二维蛋白质电泳方法应用于不同细胞周期阶段的亚历山大菌提取物，以解决翻译/翻译后调控的剩余可能性。这反过来又将导致鉴定和分离与毒素产生有关的基因。上述两种方法都将提供进行调控研究所需的遗传工具，以确定这些基因开启和关闭的机制。所获得的信息将有助于解开一些长期以来围绕着甲藻中蛤蚌毒素代谢作用的谜团，以及在不同分离物或不同环境条件下单个分离物中观察到的毒素变异性。

9415536 Anderson PROJECT ABSTRACT Blooms of toxic dinoflagellates in the genus Alexandrium result in outbreaks of paralytic shellfish poisoning (PSP), one of the more serious of the global marine phenomena collectively termed "red tides". 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, stages of fisheries species, and death of marine mammals, fish, and seabirds. These impacts are caused by the saxitoxins, a family of neurotoxins produced by the dinoflagellates and accumulated in zooplankton, shellfish, or fish during feeding. The overall objective of this project is to identify genes involved in toxin production in Alexandrium spp. and other saxitoxin-producing organisms and to study the manner in which they are regulated. The approach will use differential display to identify messenger RNA differences between cells that differ in their ability to make toxin. Differentially expressed sequences can then be used to screen for the putative toxin genes in a wide range of toxic and non-toxic species of dinoflagellates and cyanobacteria. Other indirect evidence for the toxin-specificity of the identified genes will derive from manipulations of dinoflagellate metabolism using inhibitors that block toxin biosynthesis at different levels. If the differential display approach is not successful because the toxin genes are not regulated at the transcriptional level, traditional methods of two-dimensional protein electrophoresis will be applied to extracts of Alexandrium from different cell cycle stages to address the remaining possibility of translational/post-translational regulation. This in turn will lead to the identification and isolation of genes implicated in toxin production. Both of the above approaches will provide genetic tools needed to conduct regulatory studies to determine the mechanisms by which these genes are turned on and off. The information gained will aid in deciphering some of the long-standing mysteries surrounding the metabolic role of saxitoxin in dinoflagellates and the toxin variability observed in different isolates or in individual isolates under different environmental conditions.