HORMONAL REGULATION OF ENZYME LEVELS IN MAMMALIAN TISSUE

这项研究的主要目标是了解 酶的胞质形式的基因表达 来自大鼠的β-烯醇式丙酮酸羧激酶（PEPCK）。这涉及 确定启动子调控区的功能组织 以更好地确定其急性 对cAMP、糖皮质激素和胰岛素的基因转录的反应性。 了解PEPCK基因的调控表达将提供 深入了解不同组织中代谢过程的模式， 以及调节激素的作用机制及其细胞内 信号. 这项研究的具体目标是： 1.确定所鉴定的转录调控元件的功能 在PEPCK启动子内，并将此功能与细胞内的 激素如胰高血糖素（通过cAMP起作用）、胰岛素和 糖皮质激素 2.确定启动子中负责指导 PEPCK基因在转基因小鼠肝脏和肾脏中的表达 动物这是识别跨- 作用因素，其模式的代谢特征， 特定组织 3.分离与PEPCK启动子内特定结构域结合的蛋白质 并确定它们在基因表达的激素调节中的作用。 4.使用体外转录测定来测试 转录活性蛋白质，以重建调节 确定特定蛋白质-蛋白质和蛋白质-DNA的方法 相互作用，控制PEPCK基因的转录。 我们工作的长期目标是提供一个基础， 调节系统的功能对葡萄糖稳态至关重要， 哺乳动物这项研究直接关系到我们对糖尿病的认识， 设计治疗方法来影响一种关键酶的表达， 肝和肾血管生成。这方面的进展将大大有助于 试图开发能够驱动该启动子的嵌合启动子系统， 在肝脏中特异性地调节连接的结构基因的表达; 这是一个对有效的基因治疗至关重要的系统。

The broad goal of this research is to understand the specific regulation of the expression of the gene for the cytosolic form of the enzyme P-enolpyruvate carboxy-kinase (PEPCK) from the rat. This involves determining the functional organization of the promoter-regulatory region of the gene to better define the mechanisms responsible for its acute responsiveness to gene transcription to cAMP, glucocorticoids and insulin. Understanding the regulated expression of the PEPCK gene will provide insight into the patterning of metabolic processes in different tissues as well as mechanism of action of regulatory hormones and their intracellular signals. The specific goals of this research will be to: 1.Determine the function of transcriptional regulatory elements identified within the PEPCK promoter and correlate this function with the cellular effect of hormones such as glucagon (acting via cAMP), insulin and glucocorticoids. 2.Identify sequences in the promoter which are responsible for directing the expression of the gene for PEPCK to the liver and kidney in transgenic animals. This is a necessary first step toward the identification of trans- acting factors which pattern the metabolic profile characteristic of specific tissues. 3.Isolate proteins which bind to specific domains within the PEPCK promoter and establish their role in the hormonal regulation of gene expression. 4.Using an in vitro transcription assay to test the functioning of transcriptionally active proteins in order to reconstitute the regulatory process for determining the specific protein-protein and protein-DNA interactions which control transcription of the PEPCK gene. The long-term objective of our work is to provide a basis for understanding the functioning of a regulatory system critical to glucose homeostasis in mammals. This research relates directly to our knowledge of diabetes and to the design of treatments to affect the expression of a key enzyme in hepatic and renal gluconeogenesis. Progress in this area will greatly aid attempts to develop a chimeric promoter system capable of driving the regulated expression of linked structural genes specifically in the liver; a system which is of vital importance to effective gene therapy.