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Mouse Amelogenin Exons 8 and 9: Sequence Analysis and Protein Distribution

Department of Pediatric Dentistry, Dental School, University of Texas Health Science Center San Antonio, MSC 7888, 7703 Floyd Curl Drive, San Antonio, TX 78229-3900, USA; and
¹ Department of Growth & Development, Pediatric Dentistry Division, School of Dentistry, University of California San Francisco, San Francisco, CA;

View larger version (42K): [in this window] [in a new window]   Figure 1. RT-PCR analysis of mouse amelogenin with the use of cDNA derived from a mouse tooth cDNA library and mouse immortalized dental cell lines. Two different RNA transcripts have been isolated and sequenced within the exons 2–9 mouse AMELX region (A). Lane 1, DNA markers; Lane 2, negative control; Lane 3, mouse tooth cDNA library; Lane 4, odontoblast cell line (MO6-G3); Lane 5, ameloblast cell line (MEOE-3M). DNA sequence analysis of mouse AMELX exon 8 and exon 9 and their splice variants (B). DNA sequence alignment of the mouse and rat amelogenin cDNA sequence for exons 8 & 9 showed a 93% homology between species (B). Both mouse and rat sequences contain a stop codon at position 74 (B; black rectangle). Protein prediction of the exons 8 and 9 region showed 87% homology with the predicted rat protein (C). The mouse-predicted exons 8 and 9 encoded protein sequence differs in 3 amino acids from the predicted rat protein sequence (C; black circles).

View larger version (133K): [in this window] [in a new window]   Figure 2. Immunohistochemistry of the mouse dental cell lines with amelogenin exons 8 and 9 antibody. Panels A & B show low (A) and high (B) magnification of ameloblast cells MEOE-3M. Panel C shows low magnification of odontoblast cells MO6-G3. Panel D shows negative control in MO6-G3 cells. Bars: 62.5 µm (A); 31.25 µm (C); 15.625 µm (B, D). Immunohistochemistry of mouse developing tooth organs showed the developmental pattern of amelogenin exons 8 and 9 encoded peptide. Panels E-G show positive staining within the ameloblast (AM), odontoblasts (OD), and stratum intermedium cells (SI). Pre-odontoblasts (pOD; panel E) and mature odontoblasts (panels I-M) were devoid of staining. SI staining was down-regulated (panel G, black arrow) concomitant with the maturation stage of ameloblasts (G). Note the high expression of amelogenin in secretory ameloblasts (F), in contrast to the negative expression in maturation ameloblasts (I–M). Panels I-M show intense biphasic positive staining in the enamel (E). Enamel staining is mainly observed near the dentin-enamel junction and in the newly formed matrix (panel J, black arrows). Enamel staining is clearly diminished near the cemento-enamel junction (M; black arrow). No staining was seen within the dentin (D) or dental pulp cells (DP). Bars: 62.5 µm (I); 25 µm (H,L,M); 15.625 µm (E-G); 12.5 µm (J, K). Panel H shows negative control after primary antibody omission.

Journal of Dental Research, Vol. 84, No. 7, 613-617 (2005)
DOI: 10.1177/154405910508400706


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