CHITIN BIOSYNTHESIS AND THE MOSQUITO PERITROPHIC MATRIX

蚊子传播的病原体继续对人类健康产生重大影响 世界各地的人口，疟疾和登革热 被认为是我们重新出现的两种最重要的疾病。所有病原体 通过蚊子传播的食物与血粉一起摄入，因此 他们必须在MiG-Gut环境中生存，并能够穿透 营养周基质和中肠上皮到达它们的位置 发展。尽管中肠作为决定因素的重要性 媒介能力，几乎没有关于媒介能力的具体信息 发生在体内的关键生理过程的遗传调节 吸血后的中肠。其中一个过程是 形成围绕血丸的周围性基质(PM)。 在物理上将它与中肠上皮分开。我们已经克隆了一个完整的- 埃及伊蚊谷氨酰胺合成酶(GS)基因长度的测定 它是中肠特异的，是由血液喂养引起的。我们的假设 在PM形成过程中GS对甲壳素的合成是至关重要的吗？ 谷氨酰胺：谷氨酰胺所必需的谷氨酰胺： 果糖-6-磷酸转氨酶(GFAT)催化合成 氨基葡萄糖-6-磷酸。酶分析还表明，GFAT和 几丁质合成酶(CS)在此过程中起着调节和关键作用 生化过程。因为人们对此知之甚少 参与PM甲壳素形成的生物合成途径，或遗传 对这一途径的调控，我们在这里提出了生化和分子 对GS、GFAT和CS的研究应提供对以下方面的明确理解 这一过程。具体地说，我们将(A)获得GS、GFAT、 和CS，以及核苷酸和假定的翻译产生序列 与其他生物的比较，(2)使用这些生物的cdna克隆 三种酶来评估转录活性的时间和位置， 使用Northern分析和原位杂交，并获得 用于抗体生产和后续生产的重组蛋白 免疫定位和酶抑制研究，以及(3)MAP和 对GS、GFAT和CS的基因组结构进行测序以进行鉴定 并描述了这些调控基因表达的机制。我们将使用 许多技术包括对启动子序列的广泛研究， 转基因蚊子，凝胶迁移率改变分析，DNA酶I 足迹和生理研究，以评估潜在的启动子和 增强子序列。这些基因都不是从蚊子身上分离出来的 我们预计，满足我们的具体目标将为我们提供更多 更清楚地了解与PM形成相关的甲壳素合成。一个 对中肠的生化事件有更全面的了解 在血液喂养之后也有可能提供新的方法 用来干扰病原体的传播。同样，使用 改造抗病原体蚊子的策略将 要求提供并彻底了解符合以下条件的推广人 在正确的时间和正确的地点诱导。

Mosquito-born pathogens continue to have a major impact on the health of human populations throughout the world, and malaria and dengue fever are considered two of our most important reemerging diseases. All pathogens transmitted by mosquitos are ingested with the blood meal and consequently they must survive within the mig-gut environment and be able to penetrate the peritrophic matrix and mid-gut epithelium to reach their site of development. Despite the importance of the midgut as a determinant for vector competence, little specific information is available regarding the genetic regulation of critical physiological processes that occur within the midgut following blood feeding. One of these processes is the formation of the peritrophic matrix (PM) that surrounds the blood bolus nd physically separates it from the midgut epithelium. We have cloned a full- length cDNA for Aedes aegypti glutamine synthetase (GS) and determined that it is mid-gut specific and induced by blood feeding. Our hypothesis is that GS is critical for chitin synthesis in PM formation y providing the glutamine necessary for the glutamine necessary for the glutamine: fructose-6-phosphate aminotransferase (GFAT) catalyzed production of glucosamine-6-phosphate. Enzyme assay analyses also indicate that GFAT and chitin synthase (CS) play regulatory and critical roles in this biochemical process. Because very little is known concerning the biosynthetic pathway involved in PM chitin formation, or on the genetic regulation of this pathway, we propose herein biochemical and molecular studies of GS, GFAT, and CS that should provide a cleared understanding of this process. Specifically, we will (a) obtain DNA clones for GS, GFAT, and CS, and nucleotide and putative translation production sequences will be compared with those from other organisms, (2) use cDNA clones of these three enzymes to assess timing and location of transcriptional activity, using northern analysis and in situ hybridization, and to obtain recombinant proteins for antibody production and subsequent immunolocalization and enzyme inhibition studies, and (3) map and sequence the genomic structures for GS, GFAT and CS in order to identify and characterize those mechanisms regulating gene expression. We will use a number of techniques including extensive studies of promoter sequences, transgene mosquitos, electrophoresis mobility shift assay, DNase I footprinting, and physiological studies to evaluate potential promoter and enhancer sequences. None of these genes has been isolated from mosquitos and we anticipate satisfying our specific aims will provide us with a much clearer understanding of chitin synthesis associated with PM formation. A more complete understanding of biochemical events operating in the mid-gut following blood feeding also as the potential of providing new approaches for disrupting pathogen transmission. Likewise, the goal of using transformation strategies to engineer pathogen-resistant mosquitos will require the availability and thorough understanding of promoters that are induced at the right time and at the right place.