Poly(A)+ RNA from Tetrahymena: stimulation of protein synthesis in vitro

Abstract

Cell-free synthesis of high molecular weight polypeptides, programmed by RNA from Tetrahymena pyriformis strain W is reported, and methods for preparation of the RNA are described. The RNA was extracted by the SDS-phenol-chloroform-isoamyl alcohol technic. The bulk of extracted RNA was ribosomal and on sucrose gradients peaked at approximately 17S and 25S. After heat denaturation all the 25S RNA was converted to 17S, indicating the presence of hidden breaks, possibly the result of nuclease activity during extraction. Nevertheless, when poly(A) +/- RNA was collected using oligo-(dT)-cellulose column chromatography, it promoted a 15-fold increase in incorporation of [35S] methionine into TCA-precipitable material. Slab-gel electrophoresis and autoradiography of the product revealed 12 different major polypeptides, varying in weight from 28,000 to 65,000 Daltons. A method for preparation of translatable RNA from Tetrahymena will make possible the comparison of messenger RNAs associated with specific cell structures and with different developmental events.