ALTERNATIVE SPLICING OF MOUSE AMELGENINS

牙釉质发育异常（AI）是一组异质性的遗传 主要表现为牙釉质缺陷的疾病 阵 人类X连锁AI的遗传基础是 釉原蛋白基因突变。一种复杂的釉原蛋白混合物 包括显影剂的有机组分的大约90 釉质基质 单个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 在所有物种中，选择性剪接有助于 釉质基质中釉原蛋白的多样性。 喂养老鼠 釉原蛋白转录物的比例， 特定的剪接途径在牙形成过程中发生变化。 以实现 了解釉质生物矿化的分子机制， 是鉴定各种釉原蛋白mRNA，确定其 相对丰度和表达时间，验证其 翻译产物分泌到釉质基质中， 以生物化学方式表征同种型，以深入了解功能。 本报告的具体目标是： 1. 为了分离和表征对应于这些基因的cDNA， 选择性剪接的釉原蛋白mRNA在小鼠牙胚中的表达 发展 2. 为了证明在小鼠发育的釉基质釉原蛋白 选择性剪接形成的蛋白质。 3. 为了表征选择性剪接模式的变化， 在牙发生过程中发生的釉原蛋白初级RNA转录物。 4. 目的：异源表达釉原蛋白异构体，并对其进行鉴定 关于Ca2+结合、羟基磷灰石结合及其影响 釉质微晶的种子生长。 所有小鼠釉原蛋白mRNA将被克隆和测序。 的 釉原蛋白亚型对应于这些翻译产物 mRNA将通过激光解吸质谱法、氨基酸分析法和质谱分析法表征。 酸组成分析和肽图谱。 的时空表达 不同的釉原蛋白亚型，我们将使用啮齿动物检查 门齿模型 出生后第9天的小鼠下颌切牙将 将其切成薄段，并将每种釉原蛋白的相对水平 使用半定量逆转录聚合酶测定mRNA 链反应 门牙将在组织学研究中进行检查， 通过免疫组织化学研究釉原蛋白RNA加工的变化 成釉细胞细胞分化的阶段。 釉原蛋白亚型将 异源表达，以Ca2+和羟基磷灰石为特征 结合研究及其对牙釉质种子生长的影响 微晶

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.