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  • 1
    Electronic Resource
    Electronic Resource
    Springer
    Calcified tissue international 35 (1983), S. 366-371 
    ISSN: 1432-0827
    Keywords: Enamel proteins ; Hamster ; Biosynthesis ; Enamelin ; Amelogenin
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine , Physics
    Notes: Summary Experiments were designed to detect and determine the biosynthetic behavior of enamel proteins in Syrian Golden hamsters. Enamel matrix proteins were extracted from 3-day-old postnatal first molar tooth organs. Labeling pulse/chase experiments with [35S]-methionine followed by light microscopic autoradiography, or polyacrylamide slab gel electrophoresis and fluorography, showed the synthesis of epithelial-specific gene products. Synthesis and secretion of enamel proteins required approximately 30 min under thesein vitro organ culture conditions; both enamelin and amelogenin proteins were synthesized and secreted into the forming extracellular matrix. Amelogenin proteins were secreted initially and rapidly degraded into increasingly smaller polypeptides. In contrast, enamelin proteins were secreted at a slower rate and remained more or less stable over the duration of the experiment. The specific activities of both classes of proteins increased over a 6-hour synthesis period, indicating the accumulation of both proteins into the forming extracellular matrix. Comparisons of the kinetics of formation and posttranslational processing of enamelin and amelogenin are consistent with the presence of possibly two different gene products in hamster secretory ameloblasts.
    Type of Medium: Electronic Resource
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  • 2
    ISSN: 1432-0827
    Keywords: Amelogenin ; Expression ; Enamel ; Recombinant DNA ; Tooth
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine , Physics
    Notes: Abstract A mouse cDNA encoding a 180 amino acid amelogenin was subcloned into the pET expression plasmid (Novagen, Madison, WI) for production in Escherichia coli. A simple growth and purification protocol yields 20–50 mg of 95–99% pure recombinant amelogenin from a 4.5-liter culture. This is the first heterologous expression of an enamel protein. The expressed protein was characterized by partial Edman sequencing, amino acid composition analysis, SDS-PAGE, Western blotting, laser desorption mass spectrometry, and hydroxyapatite binding. The recombinant amelogenin is 179 amino acids in length, has a molecular weight of 20,162 daltons, and hydroxyapatite binding properties similar to the porcine 173 residue amelogenin. Solubility analyses showed that the bacterially expressed protein is only sparingly soluble in the pH range of 6.4–8.0 or in solutions 20% saturated with ammonium sulfate. The purified protein was used to generate rabbit polyclonal anti-amelogenin antibodies which show specific reaction to amelogenins in both Western blot analyses of enamel extracts and in immunostaining of developing mouse molars.
    Type of Medium: Electronic Resource
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  • 3
    Electronic Resource
    Electronic Resource
    Springer
    Calcified tissue international 55 (1994), S. 302-310 
    ISSN: 1432-0827
    Keywords: Amelogenin ; Alternative splicing ; Enamel ; Tooth
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine , Physics
    Notes: Abstract A heterogeneous mixture of amelogenins can be extracted from developing tooth enamel matrix. In an attempt to discover the extent to which alternative splicing of the amelogenin primary RNA transcript can generate unique isoforms, we have conducted a thorough search for cDNAs amplified by reverse transcription-polymerase chain reaction (RT-PCR). Over 2400 colonies were screened by colony hybridization. Seven different alternatively spliced amelogenin mRNAs were isolated. The predicted translation products of the messages are 194, 180, 156, 141, 74, 59, and 44 amino acids in length. RT-PCR amplification products not predicted by these seven amelogenin cDNAs were characterized. The intron separating exons 5 and 6 was cloned and sequenced. Using rapid amplification of cDNA ends (RACE) techniques, the 5′ ends of the amelogenin mRNAs were cloned and characterized. The finding that the same exon 1 is common to all of the cloned mRNAs indicates that mouse amelogenin is transcribed from a single promoter. The mouse amelogenin transcription and translation initiation sites, the 5′ untranslated leader, and the segment encoding the signal peptide were determined. The distinctly nonamelogenin-like exon 4, first observed in human amelogenin cDNAs, has also been found in mice. Antibodies were raised to synthetic exon 4-encoded polypeptides and used to immunostain Western transfers and histologic tooth sections.
    Type of Medium: Electronic Resource
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