PORCINE ENAMEL PROTEINS

University Of Texas Hlth Sci Ctr San Ant, San Antonio TX

Investigators

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Abstract

DESCRIPTION: (Adapted from investigator's Abstract) The long term objectives are to determine the molecular mechanisms of dental enamel formation and to identify the genes responsible for amelogenesis imperfecta (AI). Four specific aims are proposed: 1) to clone and characterize cDNAs encoding porcine enamelin, the 29 kDa calcium binding protein, amelogenin, tuftelin and sheathlin; 2) to clone and characterize the porcine amelogenin gene(s), determine the number of porcine amelogenin gene(s), and their chromosomal localization(s); 3) to clone and characterize human cDNA and genomic clones encoding enamelin and the 29 kDa calcium binding protein (CBP) and to determine their chromosomal localizations; 4) to test for linkage between genes encoding enamel matrix proteins and amelogenesis imperfecta. The amino-terminal sequence of porcine enamel matrix proteins has been provided by a collaborator, Dr. Makato Fukae. The mRNA encoding these proteins are cloned and fully characterized by cloning PCR amplification products, 5' and 3' rapid amplification of cDNA ends (RACE), and by direct screening of a porcine enamel organ epithelia-specific Lambda cDNA library. The porcine amelogenin gene(s) is isolated from a male porcine genomic library. The structure of the gene is determined by the characterization of PCR amplification products, restriction analyses, and DNA sequencing. The number of amelogenin genes is determined by Southern blot analysis. The chromosomal localization is determined by fluorescent in situ hybridization. The human cDNA and genomic clones are isolated from a human tooth-specific cDNA library and a Lambda genomic library. The chromosomal localizations are determined by fluorescent in situ hybridization. Linkage analysis is performed on pedigrees displaying dominantly inherited AI using a candidate gene approach. Specific mutations are identified by single stranded conformatinal polymorphism analysis and characterized by DNA sequencing.