MOLECULAR GENETIC STUDIES

分子遗传学研究

• 批准号: 6203502
• 负责人: GERARDO Raul VASTA
• 金额: $ 24.84万
• 依托单位: UNIVERSITY OF MARYLAND BALTIMORE
• 依托单位国家: 美国
• 财政年份: 1999
• 资助国家: 美国
• 起止时间: 1999-08-01 至 2000-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6203502
• 关键词: Dinoflagellates; actins; microorganism classification; microorganism culture; microorganism genetics; microorganism toxin; mitochondrial DNA; natural gene amplification; nucleic acid probes; nucleic acid sequence; polymerase chain reaction; ribosomal RNA; subtraction hybridization; systematic biology; water environment

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.

来自马里兰州大学的科学家们最近将 暴露在切萨皮克湾的鱼类死亡中，造成严重的健康问题， 其特征在于神经系统和可能的免疫功能障碍。虽然 这些鱼类死亡的病原体尚未确定，这些 爆发类似于其他发生在北卡罗来纳州， 被认为是有毒的甲藻Pfiesteria piscicida。两 鱼类死亡和人类健康问题似乎是毒素介导的;然而， 该毒素或作用方式尚未被鉴定。隔离、 杀鱼潜蝇及其近缘种鉴定和特征 由于它们的形态相似性和 生命周期的复杂性。目前的分类法主要基于 纤维素装甲板（囊）的超微结构观察 营养无性阶段尽管有争议，但一些报道称， 超过24个生命周期阶段的P. piscicida，其中表现出 典型的双鞭毛以及变形虫形态，把它放在 恐龙变形虫目。此分类尚未得到验证， 分类学的分子模型，部分原因是专业知识和BSL 3 培养、鉴定和毒性试验所需的密封装置 不广泛使用。最近对马里兰州分离株的培养表明， 除了杀鱼类外，也有密切相关的物种， 目前的培养方法是可行的。我们建议 应用分子为他们的文化可以应用。我们建议申请 分子方法用于鉴定和分类这些 生物体基于许多基因序列，包括rRNA，mtDNA， 微卫星DNA和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PCR和 为应用甲藻基因而优化的"通用"引物。 我们已经成功地将这些技术应用于分子生物学， 相关原生生物的特征。这项工作，与 减法遗传学，将提供物种，毒素， 用于快速、灵敏和准确的阶段特异性分子探针 鉴定和定量。研究将与其他 核心设施内的研究，以培养有毒的甲藻， 与该领域的专家合作。