CLONING AND CHARACTERIZATION OF TUFTELIN GENE

Tuftelin 基因的克隆和表征

• 批准号: 3462460
• 负责人: MUHAMMAD M BASHIR
• 金额: $ 9.67万
• 依托单位: UNIVERSITY OF PENNSYLVANIA
• 依托单位国家: 美国
• 财政年份: 1993
• 资助国家: 美国
• 起止时间: 1993-04-01 至 1998-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3462460
• 关键词: RNA splicing; RNase protection assay; animal tissue; cow; dentinogenesis; embryo /fetus tissue /cell culture; enamel organ; extracellular matrix proteins; gene expression; genetic enhancer element; genetic library; genetic promoter element; genetic transcription; genetically modified animals; high performance liquid chromatography; immunocytochemistry; laboratory mouse; molecular cloning; northern blottings; nucleic acid sequence; protein sequence; protein structure function; recombinant proteins; restriction mapping; tooth enamel; western blottings

The working hypothesis of this proposal is that enamel proteins play a central role in the process of amelogenesis and that determination of the structure and expression of these proteins is essential to an understanding of that role. With presently available techniques, solubilized enamel proteins can be divided into two classes of proteins, amelogenins and enamelins, containing a total of 15 to 25 components. It is not known how many of these components represent the products of individual genes, whether the multiple components are derived from the physiologic breakdown of a limited number of proteins or are artefactually produced. The overall objective of this proposal is to characterize the structure and expression of the enamelin protein, tuftelin, and to relate the structure of the protein to its potential function. To achieve this objective, 4 Aims are proposed: (1) To complete the characterization of the bovine tuftelin gene. (2) To identify the functional elements of the tuftelin gene promoter, the site(s) of transcription initiation and whether the primary transcript is alternatively spliced. (3) To determine the temporal and spatial expression of tuftelin-related polypeptides in the developing bovine enamel organ. (4) To investigate potential structure/function relationships of tuftelin using recombinantly expressed protein. Identification of functionally significant promoter elements is essential to an understanding of control of expression of the tuftelin gene, and it is important to determine whether alternative splicing contributes to the observed hetgerogeneity of enamelins. Because of the difficulty involved in purifying native tuftelin, recombinant synthesis is the method of choice for obtaining it in quantity. The proposed studies will use state of the art molecular and cellular biology, immunologic and physical techniques. The information gained from these studies may ultimately (i) lead to better understanding of the functions of enamel proteins, (ii) lead to an explanation of some genetic defects affecting the enamel matrix, (iii) lead to the development of more effective and biologically compatible restorative materials.

该提案的工作假设是牙釉质蛋白发挥着 釉质形成过程中的核心作用及其决定 这些蛋白质的结构和表达对于 对这个角色的理解。 利用目前可用的技术， 溶解的牙釉质蛋白可分为两类蛋白质， 牙釉蛋白和牙釉蛋白，共含有15～25种成分。 它 不知道这些组件中有多少代表了以下产品： 单个基因，多个成分是否源自 有限数量的蛋白质的生理分解或 人工产生的。 该提案的总体目标是 表征牙釉质蛋白的结构和表达， tuftelin，并将蛋白质的结构与其潜力联系起来 功能。 为实现这一目标，提出 4 项目标： (1) 完成牛 tuftelin 基因的表征。 (2) 至 鉴定 tuftelin 基因启动子的功能元件 转录起始位点以及初级转录本是否是 交替拼接。 (3)确定时间和空间 发育牛中tuftelin相关多肽的表达 釉质器官。 (4) 研究潜在的结构/功能 使用重组表达蛋白的 tuftelin 关系。 鉴定具有重要功能的启动子元件至关重要 了解 tuftelin 基因表达的控制 对于确定选择性剪接是否有助于 观察到牙釉质的异质性。因为涉及的难度 在纯化天然tuftelin时，重组合成的方法是 选择大量获得它。拟议的研究将使用状态 分子和细胞生物学、免疫学和物理学领域的技术 技术。 从这些研究中获得的信息最终可能 (i) 有助于更好地了解牙釉质蛋白的功能，(ii) 解释了一些影响牙釉质的遗传缺陷 矩阵，（iii）导致开发更有效和生物学的 兼容的修复材料。