MOLECULAR GENETIC STUDIES

马里兰大学的科学家最近发现 在切萨皮克湾暴露于鱼类死亡，有严重的健康问题， 以神经学和可能的免疫功能障碍为特征。尽管 这些鱼类死亡的致病因素尚未确定，这些 疫情与北卡罗来纳州发生的其他疫情相似，并已 被归因于有毒的甲藻Pfiester piscicida。两者都有 菲斯克的杀戮和人类健康问题似乎是由毒素介导的；然而， 毒素或作用方式尚未确定。与世隔绝 稻纵卷叶蜂及其近缘种的鉴定和特性 由于它们在形态上的相似性以及 它们生命周期的复杂性。当前的分类法主要基于 黄海地区纤维素甲(囊)的超微结构观察 营养无性阶段。尽管有争议，但据报道，一些 超过24个生活史阶段的稻纵卷叶蜂，表现出 典型的双鞭毛虫以及变形虫形态，将其放置在 点一份迪诺阿米巴。此分类尚未通过以下验证 分类学的分子模型，部分原因是专业知识和BSL3 培养、鉴定和毒性分析所需的容器是 不是随处可得的。最近对马里兰州分离株的培养表明 除了Piscicida外，还存在密切相关的物种，以及 他们目前的文化方法是可以应用的。我们建议 适用于他们的培养可以应用分子。我们建议申请 鉴定和分类这些细菌的分子方法 基于许多基因序列的生物体，包括rRNA，mtDNA， 微卫星DNA，以及保守的蛋白质编码序列，如肌动蛋白。 这些序列可以通过应用聚合酶链式反应和 针对甲藻基因的应用进行了优化的“通用”引物。 我们已经成功地将这些技术应用于分子 相关原生生物的特征。这项工作与 减法遗传学，将为物种、毒素和 快速、灵敏、准确的阶段性分子探针 鉴定和量化。研究将与其他人协调 在核心设施内培养有毒甲藻的研究 与该领域的专家合作。

Scientists from the University of Maryland have recently associated exposure to fish kills on the Chesapeake Bay with serious health problems, characterized by neurologic and possible immune dysfunction. Although the causative agent of these fish kills has not been identified, these outbreaks resemble others which have occurred in North Carolina and have been attributed to the toxic dinoflagellate Pfiesteria piscicida. Both fisk kills and human health problems appear to be toxin-mediate; however, the toxin or the mode of action have not been characterized. Isolation, identification and characterization of P. piscicida and related species pose special problems due to their morphological similarity and to the complexity of their life cycles. Current taxonomy is based largely upon ultrastructure examination of cellulose armored plates (thecae) from the vegetative asexual stage. Although controversial, some of the reported greater than 24 life cycle stages of P. piscicida, which exhibits both typical biflagellated as well as ameboid morphologies, place it in the order Dinoamoebales. This classification has not been validated by molecular models of taxonomy, in part, because the expertise and BSL3 containment required for culture, identification and toxicity assays are not widely available. Recent cultures of Maryland isolates suggests that in addition to P. piscicida, closely related species are also present, and that current methodology for their culture can be applied. We propose to apply molecular for their culture can be applied. We propose to apply molecular approaches for identification and classification of these organisms based on a number of gene sequences, including rRNA, mtDNA, microsatellite DNA, and conserved protein-coding sequences such as actin. These sequences can be obtained through the application of PCR and "universal" primers optimized for the application of dinoflagellate genes. We have successfully applied these techniques for molecular characterization of related protists. This work, in conjunction with subtractive genetics, will provide the basis for species-, toxin-, and stage-specific molecular probes for rapid, sensitive, and accurate identification and quantitation. Studies will be coordinate with other research within the Core Facility for the Culture Toxic Dinoflagellates in collaboration with experts in the field.