Examining the Possible Molecular Origins for Enamel Protein Complexity

¹ The Laboratory for Developmental Biology, The University of Southern California, School of Dentistry, Gerontology Building, Room 323, Los Angeles, California 90089-0191

Malcolm L. Snead, DDS, PhD, The Laboratory for Developmental Biology, The University of Southern California, School of Dentistry, Gerontology Building, Room 323, Los Angeles California 90089-0191; (213) 743-4220

Our strategy was to examine each of the three loci capable of contributing to the observed complexity of mouse amelogenin proteins recovered from forming enamel: the genome (gene); the transcription DNA technology and a complementary DNA (cDNA) clone, pMa5-5, specific to the predominant mouse amelogenin protein. An "artificial ameloblast" was engineered based on pMa5-5 and the resulting synthetic products compared to those from authentic ameloblasts. First, the genome probably is not responsible for amelogenin complexity: Southern analysis indicates that the amelogenin gene exists as a single copy in either differentiated dental tissue or germ line tissue. Thus, ectomesenchymal-derived instructive signals for ameloblast differentiation do not lead to re-arrangement or amplification of the amelogenin gene. Next, using nucleic acid hybridization techniques, we examined messenger RNA from mouse ameloblasts. Northern analysis of authentic mRNA from mouse ameloblasts, with either the intact or 3’-end of pMa 5-5 used as the reporter molecule, indicates that only one size class of mRNA was detectable. We conclude that at the sensitivity of this assay there is no evidence for multiple mRNAs. Last, "artificial ameloblasts" were engineered so that the translation apparatus could be examined as a source of amelogenin complexity. Capped, artificial mRNAs were constructed to the pMa 5-5 template and used to program the synthesis of amelogenin polypeptides by translation in a cellfree system. When the resulting total translation products were immunoprecipitated with the rabbit anti-mouse amelogenin antibody, we observed multiple polypeptides, suggesting that the utilization of alternative start sites may also contribute to the observed complexity of amelogenin proteins, at least for artificial mRNAs translated in vitro.

Note:

We would like to thank our colleagues, Drs. A.G. Fincham, M. MacDougall, H.C. Slavkin, G. Trump, and M. Zeichner-David, for many helpful and provocative discussions. We also thank the three anonymous referees for their instructive suggestions. The illustrations and other artwork are due to the skill and dedication of Mr. Pablo Bringas, Jr., and Mr. Valentino Santos. The beta-tubulin RNA transcript was the generous gift of Dr. Steven A. Johnson (The University of Southern California), who subcloned the intact beta-tubulin cDNA clone as described and provided through the generosity of Dr. Donald Cleveland (The Johns Hopkins University). MLS is supported by a USPHS Research Career Development Award from the National Institute of Dental Research, National Institutes of Health.