Isolation and Cell-free Translation of Total Messenger RNA from Germinating Castor Bean Endosperm

Abstract

Polyadenylated RNA was isolated from the total RNA fraction extracted from the endosperm tissue of 3 day old castor bean [Ricinus communis] seedlings by affinity chromatography on oligo(dT)-cellulose. This polyadenylated RNA was efficiently translated into protein when added to a messenger RNA-dependent cell-free system derived from rabbit reticulocytes. Characterization of the translational products by electrophoresis followed by autoradiography established that numerous discrete polypeptides were formed with MW from 10,000-100,000. Immunoprecipitation in the presence of antiserum raised in rabbits against the total glyoxysomal matrix proteins showed that these proteins accounted for 15%-20% of the total translational products. Attempts to reconstitute rough endoplasmic reticulum by the addition of washed castor bean microsomal membranes to the translational system were unsuccessful; these membranes severely inhibited protein synthesis. Canine pancreatic microsomes could be added to endosperm messenger RNA-dependent reticulocyte lysates at relatively high concentrations, while still allowing significant protein synthesis.