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. 1974 May;71(5):1896–1900. doi: 10.1073/pnas.71.5.1896

Isolation and In Vitro Translation of δ-Crystallin mRNA from Embryonic Chick Lens Fibers

Peggy Zelenka 1, Joram Piatigorsky 1
PMCID: PMC388349  PMID: 4525468

Abstract

Of the protein synthesized and accumulated during differentiation of embryonic chick lens fibers, 70-80% is the tissue specific protein δ-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 δ-crystallin during development. Each lens fiber mass contained an average of 10 μg of cytoplasmic RNA; approximately 0.1 μg 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 × 105 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 δ-crystallin on sodium dodecyl sulfatepolyacrylamide-agarose gels. Comparison of the tryptic peptides of δ-crystallin with those of the in vitro products from both heterologous systems established that the lens fiber mRNA contained δ-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 δ-crystallin mRNA in the cytoplasm.

Keywords: cell-free protein synthesis, oligo(dT)-cellulose, lens crystallins, differentiation

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Selected References

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