The study of enamel gene expression during vertebrate tooth development will proceed at two different levels: (1) the major enamel proteins; and (2) the enamel structural genes. The former will involve the isolation and characterization of the major enamel proteins present in the enamel extracellular matrix from four representative vertebrate classes: Cyclostomes (hagfish), Chondrichthyes (shark), Reptilia (alligator), and Mammalia (rabbit and mouse). The applicants plan to extract enamel proteins from embryonic and fetal tooth buds by four procedures and then fractionate these proteins by gel electrophoresis. The purified proteins will be characterized by amino acid composition, determination of isoelectric point, peptide mapping, and amino acid sequence analysis. Purified proteins will be used to develop polyclonal and monoclonal antibodies, and these antibodies will then be employed in immunofluorescence microscopy, immunoprecipitation assays, and immunoautoradiography. The second aspect of this subproject will involve isolation and characterization of the mRNAs responsible for the synthesis of selected vertebrate enamel proteins. These mRNAs will be used to produce bacterial clones (via production of complementary deoxyribonucleic acid (cDNA) which will enable the subsequent characterization of specific enamel structural genes. The results will provide information and methods necessary for investigations of epithelial-mesenchymal interaction during normal and abnormal craniofacial morphogenesis. Of specific interest will be the number and structure of the enamel genes. The role of these genes in craniofacial development will be assessed from the standpoint of the localization of de novo transcription of enamel genes during epithelial determination for ameloblast differentiation and the determination of enamel gene variability during vertebrate enamel evolution.
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