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 单个釉原蛋白多肽在釉质发育中起作用， 它们的改变如何导致釉质发生紊乱，例如 发生在遗传性疾病中，即成釉不全症。 与目前 可用的技术，溶解的釉原蛋白蛋白可以被拆分， 分成10个或更多的部分。 虽然生理过程或 人为退化可能造成这种异质性，最近 我们实验室的信息表明， 异质性源于两个不同基因的转录 位于X和Y染色体上，并从选择性剪接的 主要成绩单。 为了证明这一点，我们采用了一种 免疫学和蛋白质分析方法分离和表征 使用高分辨率色谱分析某些单个组分 和电泳技术。 重点将放在确定 可选拼接消息的翻译产品，以及 已解决的组分将接受广泛的免疫学和 化学分析，以区分他们从蛋白水解处理 件. 正常人的克隆和序列分析 牙釉原蛋白基因将完成，并将对这些序列进行比较 与那些通过聚合酶链反应扩增的基因， 为了更好地了解釉质发育不全（AI）患者 分子水平上的缺陷。 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，选择的釉原蛋白多肽，包括 我们设计的正常和突变的序列， 对应于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，并制定和测试有关的作用假设， 釉原蛋白在釉质发育中的作用