Cell-free translation of messenger RNAs of embryonic tooth organs: Synthesis of the major extracellular matrix proteins

Abstract

In order to understand the regulation of embryonic mammalian enamel and dentine extracellular matrix protein synthesis, the biological activity of embryonic rabbit molar tooth organ messenger RNAs has been examined. Total RNA was extracted from 26-day embryonic tooth organs and fractionated by chromatography on oligo(dT)-cellulose. Replicate samples were fractionated on sucrose density gradients and the poly(A)-containing distribution determined using a poly(U) 3 H assay. The poly(A)-containing fractions stimulated 3 H-proline incorporation 10-fold in wheat germ cell-free extracts. Analysis of the labelled reaction products on sodium dodecyl sulphate-polyacrylamide gels revealed seven major peaks, one co-migrating with procollagen alpha chains (circa 145,000 daltons) and the others migrating slightly faster than the various extracellular matrix proteins which characterize amelogenesis and dentinogenesis. Purified collagenase digestion of the cell-free reaction products eliminated the 145,000 dalton procollagen-like polypeptide. This is the first demonstration of the isolation of embryonic tooth organ messenger RNAs and provides an experimental approach by which to study the regulation of extracellular matrix formation during tooth morphogenesis. We predict that the non-collagenous proteins synthesized in vitro represent enamel proteins, alkaline phosphatase, dentine phosphoproteins and proteins associated with proteoglycans.