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， 被认为是我们最重要的两种复发疾病。所有病原体 由蚊子传播的病毒与血餐一起被摄入， 它们必须能在胃肠道环境中生存， 围食基质和中肠上皮细胞到达它们的位置， 发展尽管中肠作为决定性因素的重要性， 矢量的能力，很少有具体的信息是关于 关键生理过程的遗传调控， 血液喂养后的中肠其中一个过程是 形成围绕血团的围食基质（PM）， 将其与中肠上皮分离。我们克隆了一个完整的- 埃及伊蚊谷氨酰胺合成酶（GS）的cDNA长度，并测定 它是中肠特异性的，由血液喂养引起。我们的假设 GS是PM形成中几丁质合成的关键， 谷氨酰胺所必需的谷氨酰胺： 果糖-6-磷酸氨基转移酶（GFAT）催化生产 葡糖胺-6-磷酸酶测定分析还表明，GFAT和 几丁质合成酶（CS）在这一过程中起着调节和关键作用 生化过程因为我们对它知之甚少， 参与PM几丁质形成的生物合成途径，或在遗传上 调节这一途径，我们在此提出生化和分子 对GS、GFAT和CS的研究，应提供对 这个过程具体而言，我们将（a）获得GS、GFAT的DNA克隆， 和CS，核苷酸和推定的翻译产生序列将 与其他生物的cDNA进行比较，（2）使用这些cDNA克隆 三种酶来评估转录活性的时间和位置， 使用北方分析和原位杂交，并获得 用于抗体生产的重组蛋白和随后的 免疫定位和酶抑制研究，和（3）映射和 对GS、GFAT和CS的基因组结构进行测序， 并描述这些调节基因表达的机制。我们将使用 许多技术包括启动子序列的广泛研究， 转基因蚊子，电泳迁移率变动分析，DNA酶I 足迹法和生理学研究来评估潜在的启动子， 增强子序列。这些基因中没有一个是从蚊子身上分离出来的 我们预计，实现我们的具体目标将为我们提供更多的 更清楚地了解与PM形成相关的几丁质合成。一 更全面地了解中肠的生化活动 在血液喂养后，也有可能提供新的方法， 来阻断病原体的传播同样，使用 改造策略，以工程抗病原体蚊子将 需要可用性和彻底了解的发起人， 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.