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Origin, Splicing, and Expression of Rodent Amelogenin Exon 8

¹ Department of Cytokine Biology,
³ Department of Immunology, Forsyth Institute &
² Department of Developmental Biology, Harvard School of Dental Medicine, Boston, MA 02115, and
⁴ Division of Aging and Geriatric Dentistry, Tohoku University Graduate School of Dentistry, Sendai, 980-8575, Japan; and
⁵ Michigan Dental Research Laboratory, Ann Arbor, MI 48108

View larger version (20K): [in a new window]   Figure 1. Relative abundance of amelogenin transcripts containing exon 7 vs. those containing exons 8 & 9 and alignments of duplicated genome region with separate alignment of exon 5 with exon 8. (A) Quantitative real-time PCR analysis of amelogenin expression in first molars extracted from mice of the indicated age. Note that amelogenin transcripts with exon 7 were significantly more abundant than transcripts with exons 8 and 9. The error bars represent the standard error of the mean from 6 separate samples. EF1'1 expression served as the internal reference gene control, so the data were plotted relative to its expression. (B) Alignment of mouse exon 4, intron 4, and exon 5 (X4&5) with mouse exon 4b, intron 4b, and exon 8 (X4b8). The first underlined sequences denote exons 4 and 4b; the second underlined sequences denote exons 5 and 8. Nucleotide differences are italicized and highlighted in bold. Note the very high sequence identity starting from before exon 4 and extending past the aligned exons 5 and 8 sequence. Of 209 nucleotides, 191 are identical (91%), and all align without gaps. (C) Alignment of mouse exon 8 amino acid sequence (mouse 8) with exon 5 sequence from the indicated species. Amino acid differences are italicized and highlighted in bold. Note that when mouse exon 8 is compared with exon 5, just 8 nucleotide differences result in the encoding of 7 different amino acids between exons 5 and 8.

View larger version (75K): [in a new window]   Figure 2. Extracted amelogenins from control vs. Mmp20 null mice. (A) Enamel proteins from the first molars of three-, five-, nine-, and 11-day-old mice were subjected to electrophoresis on SDS-PAGE gels for the assessment of differences in protein size and abundance between control (+/–, +/+) and Mmp20 null (–/ –) mice. Note that several lower MW bands are missing in the –/ – lanes when compared with bands present in the controls. Also, note that the upper bands in the control lanes became progressively weaker as development progressed, while the upper bands in the –/ – lanes also faded but remained relatively strong. (B) Western blot demonstrating that the generated exon 8 antiserum was specific for exon 8. Amelogenins from the three-day-old control mouse first molar (lane +/–), recombinant mouse amelogenin (rM179) at a concentration of 1 'g (lane, M1) or 5 'g (lane, M5), and recombinant pig amelogenin (rP172) at a concentration of 1 'g (lane, P1) or 5 'g (lane, P5) were loaded onto the gel. Western blots were performed with antiserum specific for rP172 (top panel) or for mouse exon 8 (bottom panel). Note that neither rM179 nor rP172 contained exon 8, and neither reacted with the exon 8 antiserum. Also note that although lane +/– was under-loaded in the exon 8 panel, the M and P lanes were loaded identically, and the same concentration of exon 8 antiserum was used in (B) and (C). (C) Western blot of amelogenins encoded by exon 8. Amelogenins were extracted from three-day-old control (+/–) or MMP-20 null (–/ –) mice for Western blotting with the exon 8 antiserum. Note the apparent shifting of each –/ – band to a higher molecular mass, indicating a lack of proteolytic processing. Also note a putative proteolytic cleavage product at approximately 18 kDa in the +/– lane.