Identification and Characterization of a cDNA for Mouse Ameloblastin

Abstract

Ameloblastin was first identified as one of the most abundant novel transcripts from a random screening of a rat incisor cDNA library. In situ hybridization experiments have shown ameloblastin expression to be specific to ameloblasts, with highest levels in secretory and maturation stage ameloblasts and cells of the epithelial root sheath. Ameloblastin has been identified as a candidate gene for the local hypoplastic form of autosomal dominant amelogenesis imperfecta, by virtue of it's location within the critical disease locus. The purpose of this study was to isolate a full length mouse ameloblastin cDNA and determine its temporal expression pattern during odontogenesis. A newborn mouse molar cDNA library was screened using a rat ameloblastin cDNA probe. Positive clones were confirmed by PCR analysis with ameloblastin-specific primers, and their size determined with vector-specific primers. Phage clones were rescued to phagemid using Exassist helper phage and the nucleotide sequence determined. We report here the identification of two clones, exhibiting alternative splicing of the putative open reading frame, and use of multiple polyadenylation signals. Nucleotide sequence analysis indicated a high degree of similarity to rat ameloblastin, rat amelin 1 and 2 and porcine sheathlin. Reverse transcriptase-PCR analysis using mouse first and second mandibular molar mRNA indicated initial expression at E-14. This is one day after the initial expression of tuftelin (E-13) and one day prior to that of amelogenin (E-15).