CLONING, CHARACTERIZATION, AND HETEROLOGOUS EXPRESSION OF EXON-4-CONTAINING AMELOGENIN MESSENGER-RNAS

The formation of dental enamel is dependent upon amelogenins, a family of proteins constituting most of the developing enamel matrix. Depend ing upon the species, these enamel proteins are expressed from either one or two copies of the amelogenin gene. Each gene directs the synthe sis of a variety of amelogenin isoforms through alternative splicing o f their pre-mRNA transcript(s). Before the role of amelogenins in dent al enamel formation can be better understood, one must know the isofor ms that are secreted and their biochemical properties. Previously, we cloned and characterized 7 mouse amelogenin RNA messages generated by alternative splicing. The largest amelogenin cDNA encoded a 194-residu e amelogenin isoform which was the only clone to contain the 42-nucleo tide exon 4 segment. Anti-peptide antibodies raised against the derive d translation of this exon revealed an unexpectedly diverse assortment of murine amelogenins, suggesting that additional spicing variants co uld contain the exon 4 coding region. Using exon-4-specific oligonucle otide primers, we have amplified, cloned, and characterized three diff erent amelogenin RNA messages. These messages encode amelogenin polype ptides (exclusive of signal peptides) 194, 170, and 73 amino acids in length. The isotope-averaged molecular weights for the deduced, single -phosphorylated, proteins are 21,897.1, 19,113.9, and 8176.5 Daltons, respectively. Splice-site selection for the generation of these mRNAs was identical to that of the previously characterized messages for the M180, M156, and M59 except for the inclusion of exon 4. The exon-4-co ntaining amelogenin isoforms were heterologously expressed in E. coli by means of the pET11 expression system (Novagen, Madison, WI).