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Journal of Dental Research
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Cloning, Characterization, and Heterologous Expression of Exon-4-containing Amelogenin mRNAs

C.-C. Hu

University of Texas School of Dentistry, Health Science Center at San Antonio, Department of Pediatric Dentistry, 7703 Floyd Curl Drive, San Antonio, Texas 78284-7888

O.H. Ryu

University of Texas School of Dentistry, Health Science Center at San Antonio, Department of Pediatric Dentistry, 7703 Floyd Curl Drive, San Antonio, Texas 78284-7888

Q. Qian

University of Texas School of Dentistry, Health Science Center at San Antonio, Department of Pediatric Dentistry, 7703 Floyd Curl Drive, San Antonio, Texas 78284-7888

C.H. Zhang

University of Texas School of Dentistry, Health Science Center at San Antonio, Department of Pediatric Dentistry, 7703 Floyd Curl Drive, San Antonio, Texas 78284-7888

J.P. Simmer

University of Texas School of Dentistry, Health Science Center at San Antonio, Department of Pediatric Dentistry, 7703 Floyd Curl Drive, San Antonio, Texas 78284-7888

The formation of dental enamel is dependent upon amelogenins, a family of proteins constituting most of the developing enamel matrix. Depending upon the species, these enamel proteins are expressed from either one or two copies of the amelogenin gene. Each gene directs the synthesis of a variety of amelogenin isoforms through alternative splicing of their pre-mRNA transcript(s). Before the role of amelogenins in dental enamel formation can be better understood, one must know the isoforms 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-residue amelogenin isoform which was the only clone to contain the 42-nucleotide exon 4 segment. Anti-peptide antibodies raised against the derived translation of this exon revealed an unexpectedly diverse assortment of murine amelogenins, suggesting that additional spicing variants could contain the exon 4 coding region. Using exon-4-specific oligonucleotide primers, we have amplified, cloned, and characterized three different amelogenin RNA messages. These messages encode amelogenin polypeptides (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-containing amelogenin isoforms were heterologously expressed in E. coli by means of the pET11 expression system (Novagen, Madison, WI)

Key Words: amelogenin • alternative splicing • enamel proteins • tooth.

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Journal of Dental Research, Vol. 76, No. 2, 641-647 (1997)
DOI: 10.1177/00220345970760020401
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