ENCYSTATION & CHITIN SYNTHETASE OF PARASITIC PROTOZOA

该项目的总体目标是阐明终端 蓝氏贾第鞭毛虫包囊的步骤，以及 几丁质合成酶（CS）是胞壁生物合成的关键酶。 这是分化的一个基本问题， 传播肠道疾病的特别重要， 孩子 这些目标是可行的，因为我们现在有：1） 设计了一种包囊培养基， 兰氏囊肿在体外和（2）证明发育 调节CS在G. Lamblia并开始描述和比较 入侵内阿米巴的CS。 现在我们建议：1.定义所选寄生虫的影响 以及寄主因子对G.兰氏囊蚴病 为了阐明分化的控制，并最大限度地 生物活性孢囊的产量。 2.比较体外衍生 根据以下标准，将囊肿从自然感染的囊肿中分离出来： 形态学、脱囊能力和对乳鼠的感染性。 3. 阐明几丁质合成酶的发育调控 (CS)G的活动。和侵袭内阿米巴的研究 旨在评估包囊形成刺激对CS的影响 活动 4. 纯化和表征来自两种寄生虫的CS 为了确定他们的手机位置，产品， 特性. 5. 用抗体筛选表达DNA文库 和基因探针以及CS活性，以便分离CS 结构基因，确定它们的DNA序列，并推导出 其基因产物的氨基酸序列。 6. 准备探头 并研究它们的调控。 这些探测器将 用于确定包囊形成刺激对CS的影响 基因在转录水平上的表达。 这一提议的结果应使人们对各种因素有新的认识， 控制着重要病原体的生命周期， 提供无限量的纯G蓝氏囊肿的基本和 应用研究，并帮助确定CS在其他领域的作用 医学和经济上重要的生物。

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.