CLONING, DNA-SEQUENCE, AND ALTERNATIVE SPLICING OF OPOSSUM AMELOGENINMESSENGER-RNAS

The enamel layer that covers the surfaces of teeth is thickest and mos t highly mineralized in mammals. The durability of mammalian enamel ma y have allowed for selection against the lifelong replacement of teeth that is observed in other vertebrates. Variation in enamel structure among animals is thought to be the result of evolutionary changes in t he constituents of the developing enamel matrix. In placental mammals, the principal component of this matrix is amelogenin. We have determi ned the complete primary structures of two opossum amelogenins through a combination of protein sequencing, cloning, and DNA sequencing. RNA messages were cloned that encode 202- and 57-residue amelogenins, whi ch are presumed to be expressed from the same gene but differ due to a lternative splicing of identical pre-mRNAs. Ed-nan degradation of the larger amelogenin ran for 42 cycles and yielded the sequence: IPLPPHPG HPGYINFS YEVLTPLKWYQSMMRQQYPSYGYEPM. The derived 202-residue amelogeni n, assuming that serine 16 is phosphorylated, has an isotope-averaged molecular mass of 23,023.75 Daltons and a pI of 6.2. This is the large st amelogenin yet characterized. The increase in length is due to the presence of a 30-residue tandem repeat of QP(I/M) in exon 6 in the sam e position as a similar, but shorter, repeat expressed from the bovine X-chromosome. The 57-residue amelogenin, which is known from other or ganisms as the leucine-rich amelogenin protein (LRAP), has an isotope- averaged molecular mass of 6764.75 Daltons and a pI of 5.5. The opossu m enamel protein is highly homologous to those previously characterize d in eutherians and demonstrates that amelogenins were refined structu rally prior to the metatherian/eutherian divergence between 100 and 15 0 million years ago.