Genomic/proteomic analysis of human salivary glands

人类唾液腺的基因组/蛋白质组分析

• 批准号: 6349648
• 负责人: Lawrence A. Tabak
• 金额: $ 22.18万
• 依托单位: UNIVERSITY OF ROCHESTER
• 依托单位国家: 美国
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-04-01 至 2005-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6349648
• 关键词: clinical research; functional /structural genomics; genetic library; human subject; human tissue; mass spectrometry; matrix assisted laser desorption ionization; microarray technology; parotid gland; protein purification; protein structure function; saliva; salivary glands; secretion; sequence tagged sites; xerostomias

The overall goal of Subproject 3 is to compare the expression of proteins and transcripts in normal human parotid gland with parotid tissue derived from individuals with idiopathic dry mouth. The overall hypothesis is that idiopathic dry mouth is caused by a change in the expression of key regulatory or effector proteins. To identify human parotid gland-specific proteins, in Specific Aim 1: we will create an expressed-sequence-tag [EST] data base of the human parotid gland. To accomplish this goal, we will construct a directional cDNA library from normal human adult parotid gland. To accomplish this goal, we will construct a directional cDNA library from normal human adult parotid gland tissue and will sequence the 5' end of randomly selected clones. Sequences will then be compared against available databases. We hypothesize that idiopathic dry mouth results from a change in the expression of key regulatory or effector proteins leading to dysregulation of fluid production. To test this hypothesis, in Specific Aim 2: we will compare the expression of transcripts in normal glands with those derived from individuals with idiopathic dry mouth using microarrays and direct sequencing of PCR amplified transcripts. As a complement to the EST database outlined in Specific Aim 1, we propose in Specific Aim 3, to map the spectrum of proteins which are secreted by the normal human parotid and submandibular/sublingual glands. Two dimensional gel electrophoresis will be used to separate proteins and combinations of immunoblotting, Edman degradation and matrix-assisted laser desorption/ionization time-of-flight (MALDI/TOF) mass spectrometry will be used to identify the proteins. We hypothesize that differences will be observed between this "normal" profile and one obtained using saliva collected from subjects with idiopathic dry mouth, who present with seemingly "normal" flow rates. Proteins displaying variance in their level for expression will be candidates for functional roles involved in maintaining the oral cavity in a lubricated and hydrated state. Taken together, these approaches will allow us to identify underlying defects in key regulatory or effector proteins which lead to oral dryness.

子项目 3 的总体目标是比较正常人腮腺与特发性口干个体的腮腺组织中蛋白质和转录本的表达。总体假设是特发性口干是由关键调节蛋白或效应蛋白表达的变化引起的。为了识别人类腮腺特异性蛋白质，在具体目标 1 中：我们将创建人类腮腺的表达序列标签 [EST] 数据库。为了实现这一目标，我们将从正常成人腮腺构建定向 cDNA 文库。为了实现这一目标，我们将从正常成人腮腺组织构建定向 cDNA 文库，并对随机选择的克隆的 5' 端进行测序。然后将序列与可用数据库进行比较。我们假设特发性口干是由于关键调节蛋白或效应蛋白的表达变化导致液体产生失调所致。为了检验这一假设，在具体目标 2 中：我们将使用微阵列和 PCR 扩增转录本的直接测序，比较正常腺体中转录本的表达与特发性口干个体的转录本表达。作为对特定目标 1 中概述的 EST 数据库的补充，我们在特定目标 3 中建议绘制正常人腮腺和下颌下/舌下腺分泌的蛋白质谱图。二维凝胶电泳将用于分离蛋白质，免疫印迹、Edman 降解和基质辅助激光解吸/电离飞行时间 (MALDI/TOF) 质谱法的组合将用于鉴定蛋白质。我们假设这种“正常”特征与使用从特发性口干受试者采集的唾液获得的特征之间会观察到差异，这些受试者表现出看似“正常”的流速。在表达水平上表现出差异的蛋白质将成为参与维持口腔润滑和水合状态的功能角色的候选者。总而言之，这些方法将使我们能够识别导致口腔干燥的关键调节蛋白或效应蛋白的潜在缺陷。