MOLECULAR CLONING, EXPRESSION AND STRUCTURE OF ENAMEL PROTEINS

本研究提案的总体目标是确定角色(S) 个别釉原蛋白多肽在釉质发育中发挥作用，以及 它们的改变如何导致错乱的成釉作用，例如 发生于遗传性疾病，釉质发育不全。与目前 现有的技术，溶解的釉原蛋白可以被分解 分成10个或更多个组件。尽管生理过程或 最近，人工因素的退化可能是这种异质性的原因 来自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，并来自于 主要的成绩单。为了证明这一点，我们采用了一种联合 免疫学和蛋白质分析方法分离和鉴定 部分单体成分采用高分辨率层析 和电泳法。重点将放在确定 交替拼接消息的翻译产品，以及 被分解的成分将受到广泛的免疫学和 化学分析以区分它们与蛋白水解物 组件。正常人基因的克隆和序列分析 将完成釉原蛋白基因，并对这些序列进行比较 通过聚合酶链式反应扩增的基因，在 对成釉发育不全(AI)患者的认识 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 在转基因小鼠身上。最后，精选釉原蛋白多肽，包括 我们设计的正常序列和突变序列以及多肽 与在AI患者中发现的基因相对应，将通过 重组DNA技术，以评估个体的功能 多肽，并提出和测试有关的作用假说 釉原蛋白在釉质发育中的作用

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.