Isolation and characterization of polysomes and polyadenylated polysomal RNA from vicia faba meristematic root cells

Abstract

Undegraded Vicia faba polysomes from meristematic root cells were obtained after homogenization in a medium of low ionic strength provided that the pH was equal to 9.0. By minimizing the shearing forces during the homogenization step, polysomes were obtained free of mitochondrial and nuclear contaminants, measured by differential spectrophotometry and CsCl gradient centrifugation respectively. Poly(A)-containing RNA was obtained by poly(U)-Sepharose chromatography and shown to be virtually free of rRNA and its average size was 13-15 S. Approximately 9% of the purified preparation was annealed by [3H]-poly(U). Sucrose gradient analysis under denaturing conditions showed that the poly(A)-CONtaining RNA were non-degraded. This RNA was used to direct the synthesis of proteins in a heterologous cell-free system from wheat germ.