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.

subproject 3的总体目标是比较正常人腮腺中蛋白质和转录本的表达，并比较源自特发干口腔的个体的腮腺组织。总体假设是特发性干口是由关键调节或效应蛋白表达的变化引起的。为了鉴定人腮腺特异性蛋白质，在特定的目的1中：我们将创建一个人腮腺的表达序列标记数据库。为了实现这一目标，我们将从正常的人腮腺中构建一个定向cDNA文库。为了实现这一目标，我们将从正常的人腮腺组织中构建一个定向cDNA文库，并将对随机选择的克隆的5'末端进行测序。然后将将序列与可用数据库进行比较。我们假设特发性干口腔是由于关键调节或效应蛋白表达的变化导致流体产生失调的失调。为了检验这一假设，在特定的目标2中：我们将比较正常腺体中的转录本的表达与使用微阵列的特发性干嘴的个体衍生出的转录本和PCR扩增的转录本的直接测序。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中提出了绘制蛋白质的谱，这些蛋白质由正常的人腮腺和下颌/舌下腺体分泌。二维凝胶电泳将用于分离免疫印迹，Edman降解和基质辅助激光解吸/电离飞行时间（MALDI/TOF）质谱法的组合来识别蛋白质。我们假设将观察到这种“正常”特征与使用从特发性干嘴的受试者收集的唾液获得的差异，这些唾液的流量似乎是“正常”的流速。在表达水平上显示方差的蛋白质将是候选在润滑和水合状态下维持口腔的功能作用的候选。综上所述，这些方法将使我们能够识别导致口服干燥的关键调节或效应蛋白中的潜在缺陷。