Amelogenesis Imperfecta (AI) is a collection of genetic disorders primarily affecting enamel formation. Based upon phenotype and mode of inheritance approximately 12 genetic aberrations can lead to AI, while only a single locus has yet been implicated in its etiology. Recent data indicates the presence of novel enamel proteins with apparent molecular weights of 18-20 kDa (Simmer et al. Calc. Tissue Int. in press). This research proposal focuses on isolating and partially characterizing these proteins, and cloning their mRNAs. The results of this study will be essential for an RO1 application seeking to further investigate these non amelogenin enamel proteins, their genes, and their linkage to amelogenesis imperfecta. With the objective to characterize proteins that function primarily during enamel formation, the following 2 specific aims are proposed: 1. To isolate and characterize mouse non amelogenin matrix protein(s) with apparent molecular weights of 18,000 to 20,000. 2. To clone and sequence the mRNA(s) encoding these proteins. The proposed research plan accomplishes these specific aims by purifying the 18-20 kDa non amelogenin enamel matrix protein(s) and characterizing them by SDS-PAGE, Western blotting, amino acid composition determination, mass spectrometry, and amino-terminal sequencing. The purified protein(s) will be used as antigens to generate rabbit polyclonal antibodies. A mouse enamel organ epithelia-derived cDNA expression library will be constructed and screened with these antibodies. The isolated cDNA clones will be characterized by DNA sequencing. That the cloned mRNAs encode the isolated proteins is established by comparing the physical properties of the proteins to those predicted by analysis of the cDNA sequences.
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