Isolation and in vitro translation of delta-crystallin mRNA from embryonic chick lens fibers

Abstract

Of the protein synthesized and accumulated during differentiation of embryonic chick lens fibers, 70-80% is the tissue specific protein delta-crystallin. We have isolated and partially characterized the total cytoplasmic mRNA from purified lens fibers of 15-day-old embryos as an initial step toward understanding the regulated expression of delta-crystallin during development. Each lens fiber mass contained an average of 10 mug of cytoplasmic RNA; approximately 0.1 mug per fiber mass was recovered in the mRNA fraction by oligo(dT)-cellulose chromatography. The mRNA electrophoresed primarily as a single peak on a polyacrylamide-agarose gel with an apparent molecular weight of about 9 x 10(5) estimated by comparison with 28S and 18S rRNA markers. Of the protein synthesized in response to the mRNA in cell-free systems derived from Krebs II ascites tumor cells or rabbit reticulocytes, 70-80% comigrated with delta-crystallin on sodium dodecyl sulfatepolyacrylamide-agarose gels. Comparison of the tryptic peptides of delta-crystallin with those of the in vitro products from both heterologous systems established that the lens fiber mRNA contained delta-crystallin mRNA, and that no other functional mRNAs were present in detectable quantities. Thus, the specialization of protein synthesis in embryonic chick lens fibers apparently results from an accumulation of delta-crystallin mRNA in the cytoplasm.