ALTERNATIVE SPLICING OF MOUSE AMELGENINS

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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.