MOLECULAR CLONING, EXPRESSION AND STRUCTURE OF ENAMEL PROTEINS

牙釉质蛋白的分子克隆、表达和结构

• 批准号: 6270293
• 负责人: JOEL ROSENBLOOM
• 金额: $ 26.67万
• 依托单位: UNIVERSITY OF PENNSYLVANIA
• 依托单位国家: 美国
• 财政年份: 1997
• 资助国家: 美国
• 起止时间: 1997-12-01 至 2000-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6270293
• 关键词: RNA splicing; affinity chromatography; amelogenin; animal tissue; circular dichroism; conformation; dental development; dental disorder; fluorescence spectrometry; gene deletion mutation; gene expression; genetic disorder; genetic polymorphism; genetic regulatory element; genetically modified animals; human genetic material tag; human tissue; hydroxyapatites; laboratory mouse; laboratory rabbit; molecular cloning; nucleic acid sequence; point mutation; protein structure function; proteins; sex chromosomes; sex linked trait; site directed mutagenesis; southern blotting; western blottings

The overall goals of this research proposal are to determine the role(s) that individual amelogenin polypeptides play in enamel development, and how alterations in them may result in deranged amelogenesis, such as occurs in the heritable disease, Amelogenesis Imperfecta. With presently available techniques, solubilized amelogenin proteins can be resolved into 10 or more components. Although physiologic processing or artefactual degradation probably contribute to this heterogeneity, recent information from our laboratory suggests that at least some of the heterogeneity arises from the transcription of two divergent genes located on the X and Y chromosomes and from alternative splicing of the primary transcripts. To prove this, we have adopted a combined immunologic and protein analytic approach to isolate and characterize some of the individual components using high resolution chromatographic and electrophoretic techniques. Emphasis will be placed on identifying the translation products of alternatively spliced messages, and the resolved components will be subjected to extensive immunologic and chemical analyses to distinguish them from proteolytically processed components. the cloning and sequence analysis of the normal human amelogenin genes will be completed, and these sequences will be compared to those of genes, amplified by the polymerase chain reaction, in patients with Amelogenesis Imperfecta (AI) in order to better understand the defects involved at the molecular level. In addition, we plan to compare the functional characteristics of the normal and abnormal genes in transgenic mice. Finally, selected amelogenin polypeptides, including normal and mutated sequences that we design as well as peptides corresponding to those found in AI patients, will be expressed by recombinant DNA techniques, in order to evaluate functions of individual peptides, and to formulate and test hypotheses about the roles of amelogenins in enamel development.

本研究提案的总体目标是确定角色 单个釉原蛋白多肽在牙釉质发育中发挥作用，并且 它们的改变如何导致釉质生成紊乱，例如 发生在遗传性疾病，釉质生成不全症中。 与目前 可用技术，可溶解溶解的牙釉蛋白 分成 10 个或更多组件。 尽管生理加工或 人工退化可能导致这种异质性，最近 我们实验室的信息表明，至少有一些 异质性源于两个不同基因的转录 位于 X 和 Y 染色体上，并且来自可变剪接 主要转录本。 为了证明这一点，我们采用了组合 免疫学和蛋白质分析方法来分离和表征 使用高分辨率色谱分析某些单独的成分 和电泳技术。 重点将放在识别 选择性剪接消息的翻译产物，以及 解析的成分将受到广泛的免疫学和 化学分析以将其与蛋白水解加工物区分开来 成分。 正常人的克隆和序列分析 釉原蛋白基因将会完成，并且这些序列将会被比较 那些通过聚合酶链式反应扩增的基因， 患有釉质生成不全（AI）的患者，以便更好地了解 涉及分子水平的缺陷。 此外，我们计划 比较正常和异常基因的功能特征 在转基因小鼠中。 最后，选定的牙釉蛋白多肽，包括 我们设计的正常和突变序列以及肽 对应于在 AI 患者中发现的那些，将表示为 重组DNA技术，以评估个体的功能 肽，并制定和测试有关其作用的假设 釉原蛋白在牙釉质发育中的作用。