The distribution of 32P in ribonucleic acid from nucleate and anucleate half cells of Amoeba proteus

Abstract

Nucleate and anucleate half cells of Amoeba proteus were labelled with [ 32P]orthophosphate and the distribution of 32P among the labelled nucleotides was examined. Both nucleate and anucleate half cells were capable of incorporating orthophosphate into RNA. The anucleate half cell, however, incorporated 32P at a slower rate than did the nucleate half cell and there was a diminution in the relative rate of 32P incorporation into the anucleate half cell with time after enucleation. Irrespective of the rate of 32P incorporation the distribution of 32P label among the nucleotides of RNA following alkaline degradation was not significantly different between the ribonucleotides in anucleate and nucleate half cells. Several methods of RNA preparation demonstrated that the orthophosphate incorporation was in fact attributable to a polyribonucleotide. It was further shown that the radioactivity was primarily associated with 2′,3′-nucleotides following alkaline degradation of the labelled RNA, ruling out the possibility of a nucleotide-binding phenomenon. Filtration experiments suggested that the labelled cytoplasmic RNA was not bound to stable particles of a diameter greater than 50 mμ. Finally the half cells were fractionated by centrifugation and the distribution of 32P in RNA of these fractions was analyzed. The fractionation experiments led us to conclude that RNA associated with the microsomes contains much of the label in nucleate and anucleate half cells. The RNA of the microsomes has similar distributions of 32P in both nucleate and anucleate half cells and these distributions were similar to the total half-cell distributions of 32P. The soluble fractions of both half cells had distinctly different distributions of 32P from the microsomal fractions and the soluble fractions of nucleate and anucleate half cells were different from each other.