ENCYSTATION & CHITIN SYNTHETASE OF PARASITIC PROTOZOA

成囊站

• 批准号: 3137167
• 负责人: FRANCES D. GILLIN
• 金额: $ 30.61万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN DIEGO
• 依托单位国家: 美国
• 财政年份: 1988
• 资助国家: 美国
• 起止时间: 1988-04-01 至 1993-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3137167
• 关键词: Giardia; cell differentiation; chitin; complementary DNA; developmental genetics; enzyme mechanism; feces analysis; gastrointestinal hormones; gel electrophoresis; gene expression; genetic manipulation; genetic transcription; hormone regulation /control mechanism; immunofluorescence technique; immunological substance; immunoprecipitation; laboratory mouse; laboratory rabbit; ligase; microorganism culture; microorganism genetics; mutagen testing; nucleic acid hybridization; nucleic acid probes; nucleic acid sequence; phase contrast microscopy; protein biosynthesis; protein sequence; protozoal cyst; protozoal infection; structural genes

The overall goals of this project are to elucidate the terminal steps of encystation by Giardia lamblia, and the regulation of chitin synthetaase (CS), a key enzyme in cyst wall biosynthesis. This is a basic problem in differentiation and relevant to transmission of an enteric disease of particular importance in children. These goals are feasible because we have now: 1) devised an encystation medium that induces large number of G lamblia cysts in vitro and (2) demonstrated developmentally regulated CS in G. lamblia and begun to characterize and compare it to the CS of Entamoeba invadens. Now we propose to: 1. Define the influence of selected parasite and host factors on the terminal steps of G. lamblia encystation, in order to elucidate the control of differentiation and maximize the yield of biologically active cysts. 2. Compare in vitro derived cysts to cysts from natural infections by the following criteria: morphology, ability to excyst, and infectivity for suckling mice. 3. Elucidate the developmental regulation of chitin synthetase (CS) activities of G. lamblia and Entamoeba invadens by studies designed to assess the effects of encystation stimuli on CS activity. 4. Purify and characterize the CS from both parasites in order to determine their cellular locations, products, and properties. 5. Screen expression DNA libraries with antibodies and gene probes and for CS activity in order to isolate the CS structural genes, determine their DNA sequences and deduce the amino acid sequences of their gene products. 6. Prepare probes for the cloned genes and study their regulation. These probes will be used to determine the effects of encystation stimuli on CS gene expression at the transcriptional level. The results of this proposal should yield new insights into factors that control the life cycle of an important pathogen, as well as provide unlimited supplies of pure G. lamblia cysts for basic and applied research, and help define the roles of CS in other medically and economically important organisms.

该项目的总体目标是阐明终端 贾第鞭毛虫形成包囊的步骤，以及 几丁质合成酶（CS），囊壁生物合成的关键酶。 这是微分法中的一个基本问题，与 一种特别重要的肠道疾病的传播 孩子们。 这些目标是可行的，因为我们现在拥有：1) 设计了一种诱导大量 G 的包囊培养基 体外兰布里亚囊肿和（2）发育证明 在 G. Lamplia 中调节 CS 并开始表征和比较 它是内阿米巴入侵者的CS。 现在我们建议： 1. 定义所选寄生虫的影响 和宿主因素对 G. Lamplia 包囊的最终步骤的影响， 为了阐明分化的控制并最大化 具有生物活性的包囊产量。 2. 体外衍生的比较 根据以下标准将囊肿转变为自然感染引起的囊肿： 乳鼠的形态、排出能力和感染性。 3. 阐明几丁质合成酶的发育调控 通过研究G.兰伯利亚和内阿米巴入侵的（CS）活动 旨在评估成囊刺激对 CS 的影响 活动。 4. 纯化并表征两种寄生虫的 CS 以确定他们的蜂窝位置、产品和 特性。 5. 用抗体筛选表达 DNA 文库 和基因探针并用于CS活性以分离CS 结构基因，确定其 DNA 序列并推断 其基因产物的氨基酸序列。 6. 准备探针 寻找克隆的基因并研究它们的调控。 这些探针将 用于确定成囊刺激对 CS 的影响 转录水平上的基因表达。 该提案的结果应该会对因素产生新的见解 控制重要病原体的生命周期，以及 为基本和 应用研究，并帮助定义计算机科学在其他领域的作用 医学和经济上重要的生物。