Isolation and characterization of mRNA from Paramecium aurelia

Abstract

Total cellular RNA was isolated from the ciliate protozoan Paramecium aurelia by pH 9.5 chloroform octanol extraction. Passage of this RNA through an oligo(dT)-cellulose column in 0.5 M NaCl resulted in 2–3% binding, indicating the presence of polyadenylic acid sequences. These polyadenylic acid regions were estimated to be 250–500 nucleotides in length, based on their resistance to ribonuclease degradation. The oligo(dT)-cellulose bound RNA sedimented at 14–25 S in sodium dodecyl sulphate sucrose gradients. The base composition of this RNA is similar to the base composition of the DNA. This RNA was also actively translated into protein by an in vitro protein synthesizing system isolated from wheat germ. Translation was optimal under conditions similar to those used for mammalian mRNA translation. In addition, translation of the P. aurelia oligo(dT)-cellulose bound RNA was inhibited 80% by the analog 7-methylguanosine-5′-phosphate, suggesting the presence of a 5′-capped terminus.