ALTERNATIVE SPLICING OF MOUSE AMELGENINS

小鼠釉蛋白的替代剪接

• 批准号: 2015128
• 负责人: JAMES P SIMMER
• 金额: $ 10.01万
• 依托单位: UNIVERSITY OF TEXAS HLTH SCIENCE CENTER
• 依托单位国家: 美国
• 财政年份: 1995
• 资助国家: 美国
• 起止时间: 1995-01-01 至 1999-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2015128
• 关键词: RNA splicing; ameloblasts; amelogenin; calcium binding protein; cell differentiation; complementary DNA; dental development; epithelium; extracellular matrix proteins; gene expression; high performance liquid chromatography; hydroxyapatites; immunocytochemistry; in situ hybridization; laboratory mouse; mass spectrometry; messenger RNA; normal ossification; polymerase chain reaction; protein isoforms; protein sequence; protein structure function

Amelogenesis imperfecta (AI) is a heterogeneous group of genetic disorders that are manifested primarily as defects in dental enamel formation. The genetic basis for a human X-linked form of AI is a mutation in the amelogenin gene. A complex mixture of amelogenins comprise approximately 90% of the organic component of the developing enamel matrix. The molecular structures of individual amelogenin proteins are unknown as is the biological relevance of amelogenin heterogeneity. In all species examined alternative splicing contributes to the diversity of amelogenins in the enamel matrix. In mice the proportion of amelogenin transcripts that are processed through a particular splicing pathway changes during odontogenesis. To achieve an understanding of the molecular mechanisms of enamel biomineralization it is necessary to identify the assorted amelogenin mRNAs, determine their relative abundance and timing of expression, verify that their translation products are secreted into the enamel matrix, and to characterize the isoforms biochemically to gain insights into function. The Specific Aims of this report are: 1. To isolate and characterize cDNAs that correspond to the alternatively spliced amelogenin mRNAs expressed during mouse tooth development. 2. To demonstrate in the murine developing enamel matrix amelogenin proteins formed as a consequence of alternative splicing. 3. To characterize changes in the pattern of alternative splicing of the amelogenin primary RNA transcript that occur during odontogenesis. 4. To heterologously express amelogenin isoforms and characterize them with respect to Ca2+ binding, hydroxyapatite binding, and their effects on the seeded growth of enamel crystallites. All of the mouse amelogenin mRNAs will be cloned and sequenced. The amelogenin isoforms corresponding to the translation products of these mRNAs will be characterized by laser desorption mass spectrometry, amino acid composition analysis, and peptide mapping. The temporal expression of the different amelogenin isoforms will we examined using the rodent incisor model. Postnatal day 9 mouse mandibular incisors will be sectioned into thin segments and the relative levels of each amelogenin mRNA determined using semi-quantitative reverse transcription--polymerase chain reaction. The incisors will be examined in histology studies and by immunohistochemistry to correlate changes in amelogenin RNA processing to stages of ameloblast cytodifferentiation. Amelogenin isoforms will be heterologously expressed and characterized by Ca2+ and hydroxyapatite binding studies and for their effects on the seeded growth of enamel crystallites.

釉质发生不全是一组异质性的遗传病 主要表现为牙釉质缺陷的疾病 队形。人类X连锁人工智能的遗传基础是一种 釉原蛋白基因突变。一种复杂的釉质原蛋白混合物 包括显影剂中约90%的有机成分 釉质基质。个别釉原蛋白的分子结构 蛋白质是未知的，釉原蛋白的生物学相关性也是未知的。 异质性。在所研究的所有物种中，选择性剪接都有贡献 釉质基质中釉原蛋白的多样性。在小鼠中， 釉原蛋白转录本通过 在牙齿发育过程中，特定的剪接途径发生了变化。要实现 牙釉质生物矿化的分子机制研究 是鉴定各种釉原蛋白mRNAs所必需的，确定其 相对丰度和表达时间，验证它们的 翻译产物被分泌到牙釉质基质中，并 从生物化学的角度对这些异构体进行表征，以深入了解它们的功能。 本报告的具体目标是： 1.分离和鉴定与该基因对应的cDNA 小鼠牙齿表达选择性剪接的釉原蛋白mRNAs 发展。 2.在发育中的小鼠牙釉质基质中展示釉原蛋白 作为选择性剪接的结果而形成的蛋白质。 3.表征可选剪接模式的变化 釉原蛋白在牙齿发育过程中的初级RNA转录本。 4.异源表达釉原蛋白亚型并鉴定 关于钙离子结合、羟基磷灰石结合及其影响 关于釉质微晶的种子生长。 所有小鼠釉原蛋白的mRNAs都将被克隆和测序。这个 与这些基因的翻译产物相对应的釉原蛋白亚型 MRNAs将用激光解吸质谱表征，氨基 酸组成分析和多肽图谱。时间表达 对于不同的釉原蛋白亚型，我们将使用啮齿类动物进行检测 门牙模型。出生后第9天小鼠下切牙将被 切成细段和每种釉原蛋白的相对水平 半定量逆转录聚合酶检测mRNA 连锁反应。门牙将在组织学研究和 用免疫组织化学方法研究釉原蛋白RNA加工过程中的变化 进入成釉细胞分化阶段。釉原蛋白亚型将 用钙离子和羟基磷灰石异源表达和表征 牙釉质结合力研究及其对牙釉质种植生长的影响 微晶。