REGULATION OF SALIVARY GLAND-SPECIFIC GENE EXPRESSION

唾液腺特异性基因表达的调节

• 批准号: 2131375
• 负责人: Lawrence A. Tabak
• 金额: $ 19.8万
• 依托单位: UNIVERSITY OF ROCHESTER
• 依托单位国家: 美国
• 财政年份: 1993
• 资助国家: 美国
• 起止时间: 1993-02-01 至 1998-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2131375
• 关键词: DNA binding protein; DNA footprinting; affinity chromatography; clone cells; crosslink; exocytosis; gel mobility shift assay; gene deletion mutation; gene expression; genetic library; genetic mapping; genetic regulatory element; genetically modified animals; ion exchange chromatography; isoproterenol; laboratory mouse; laboratory rat; nuclear runoff assay; nucleolus; nucleoproteins; protein biosynthesis; protein purification; salivary glands; submandibular gland; transcription factor

Salivary proteins play a crucial role in protecting the hard and soft tissues of the mouth from disease. Unfortunately, little is known about the basic molecular events which regulate the synthesis of salivary gland-specific proteins. Thus, the long term goal of this program is to define the mechanisms which regulate salivary gland-specific gene expression. We have chosen as a model the gene which encodes the rat salivary glutamine/glutamic acid-rich protein-Ca (GRP-Ca), which is a member of the superfamily of proline-rich proteins. Compelling evidence points to the importance of transcriptional control in regulating the expression of tissue-specific gene products. We hypothesize that there is a set of unique cis-acting elements and transcription factors which are responsible for the expression of GRP-Ca. To test this hypothesis we propose to: (1) functionally map the cis- acting elements of the GRP-Ca gene using transgenic mice and cell lines of salivary gland and non-salivary gland origin, and (2) identify salivary gland nuclear proteins which interact with these cis-acting elements by mobility shift and DNase I foot-printing assays. These salivary gland DNA binding proteins will be purified by combinations of ion-exchange and oligonucleotide-affinity chromatography. Superimposed over the level of gene regulation afforded by the interaction of transcription factors with their cognate cis-acting elements are the mechanisms which regulate the expression of the transcription factors themselves. In salivary glands we hypothesize that transcription factor expression is controlled, in part, by secretagogues, thereby coupling exocytosis and protein biosynthesis. To test this hypothesis, we propose to measure the effect of the secretagogue isoproterenol on the transcription of the GRP-Ca gene by performing nuclear run-off assays. In addition, we will determine if isoproterenol influences the stability of the GRP-Ca transcript. Collectively, our studies will provide the fundamental information required to begin development of novel gene therapy approaches for the treatment of salivary gland hypofunction.

唾液蛋白在保护硬和软的方面起着至关重要的作用 疾病的嘴巴组织。 不幸的是，对 调节唾液合成的基本分子事件 腺体特异性蛋白质。 因此，该计划的长期目标是 定义调节唾液腺特异性基因的机制 表达。 我们选择了编码大鼠的基因的模型 唾液谷氨酰胺/谷氨酸富含蛋白-CA（GRP-CA），这是一个 富含脯氨酸的蛋白质的超家族成员。 令人信服的证据指出了转录控制的重要性 在调节组织特异性基因产物的表达时。 我们 假设有一组独特的顺式作用元素 导致GRP-CA表达的转录因子。 为了检验该假设，我们提出：（1）功能绘制顺式映射 使用转基因小鼠和细胞系的GRP-CA基因的作用元素 唾液腺和非平腺的起源，（2）识别 与这些顺式作用相互作用的唾液腺核蛋白 移动性转移和DNase I脚印测定的元素。 这些 唾液腺DNA结合蛋白将通过组合纯化 离子交换和寡核苷酸 - 亲和力色谱。 叠加于由 转录因子的相互作用与它们的同源顺式作用 元素是调节表达的机制 转录因子本身。 在唾液腺中，我们假设 转录因子的表达部分由促分子控制 从而耦合胞吐作用和蛋白质生物合成。 测试这个 假设，我们建议衡量秘密的效果 通过执行GRP-CA基因转录的异丙肾上腺素 核径流测定法。 另外，我们将确定异丙肾上腺素是否 影响GRP-CA转录本的稳定性。 总体而言，我们的 研究将提供开始所需的基本信息 开发新型基因治疗方法用于治疗 唾液腺功能低下。