REGULATION OF SALIVARY GLAND-SPECIFIC GENE EXPRESSION

唾液腺特异性基因表达的调节

• 批准号: 3223972
• 负责人: Lawrence A. Tabak
• 金额: $ 19.04万
• 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/3223972
• 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.

唾液蛋白在保护硬组织和软组织中发挥着至关重要的作用 口腔组织免受疾病侵害。 不幸的是，人们对此知之甚少 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。 我们选择编码老鼠的基因作为模型 唾液中富含谷氨酰胺/谷氨酸的蛋白钙 (GRP-Ca) 富含脯氨酸的蛋白质超家族的成员。 令人信服的证据表明转录控制的重要性 调节组织特异性基因产物的表达。 我们 假设存在一组独特的顺式作用元件，并且 负责 GRP-Ca 表达的转录因子。 为了检验这个假设，我们建议：（1）功能性地映射顺式- 使用转基因小鼠和细胞系研究 GRP-Ca 基因的作用元件 唾液腺和非唾液腺来源，以及（2）识别 与这些顺式作用相互作用的唾液腺核蛋白 通过迁移率变化和 DNase I 足迹分析来分析元素。 这些 唾液腺 DNA 结合蛋白将通过以下组合进行纯化 离子交换和寡核苷酸亲和层析。 叠加在基因调控水平上 转录因子与其同源顺式作用的相互作用 元件是调节表达的机制 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泌素控制， 从而耦合胞吐作用和蛋白质生物合成。 为了测试这个 假设，我们建议测量促分泌素的效果 异丙肾上腺素对 GRP-Ca 基因转录的影响 核径流分析。 此外，我们将确定异丙肾上腺素是否 影响 GRP-Ca 转录物的稳定性。 总的来说，我们的 研究将提供开始所需的基本信息 开发新的基因治疗方法来治疗 唾液腺功能减退。