DERIVED PROTEIN AND CDNA SEQUENCES OF HAMSTER AMELOGENIN

Hamster enamel protein extracts were analyzed by RP-HPLC and the isola ted fractions by SDS-and Western blotting using polyclonal antibodies against recombinant mouse amelogenin and anti-peptide antibodies again st the mouse exon 4-encoded sequence. Total RNA was extracted from ena mel organ epithelia and, using a 3' rapid amplification of cDNA ends ( 3' RACE) technique, the coding regions for three different amelogenin isoforms were cloned along with the 3' non-coding region. DNA sequenci ng revealed that the hamster amelogenin isoforms are 180, 73 and 59 am ino acids in length, respectively. The 59-residue amelogenin correspon ds to the leucine-rich amelogenin protein (LRAP), the 73-residue amelo genin corresponds to LRAP with the inclusion of the exon 4-encoded seq uence, while the 180-residue amelogenin is the most abundant amelogeni n isoform. Edman degradation was performed on purified hamster ameloge nin, which provided the amino acid sequence in the region encoded by t he 5' PCR amplification primer used in cloning. Therefore, tile entire derived amino acid sequence of hamster amelogenin was revealed. The h amster amelogenin amino acid sequence was aligned with all its known h omologues. Hamster differs from rat and mouse amelogenin at only three amino acid positions. Southern blot analysis using a panel of restric tion enzymes gave the same pattern for hamster DNA obtained from males and females; suggesting that in hamster, as in mouse, amelogenin is e xpressed from a single gene located on the X chromosome.