Translation of Precursors of C4 and Albumin by Isolated Guinea Pig m-RNA

Guinea pig RNA was isolated from liver homogenates by phenol-chloroform-ethanol extraction and applied to a Sigmacell type 50 column in Tris (40 mM) buffer containing KCl (1.0 M) and MgCl₂ (0.4 mM). Messenger RNA was eluted from the column, precipitated in ethanol and NaCl (1.0 M) washed in the same solution and redissolved in distilled water. The m-RNA was then added to a mixture containing rabbit reticulocyte polysomes (in some experiments pretreated with a micrococcal nuclease) an energy source, rat t-RNA, hemin, ³H-labeled leucine, 19 unlabeled amino acids and optimal concentrations of K⁺ and Mg⁺⁺. Total protein synthesis by micrococcal nuclease-treated reticulocyte lysate was increased nearly four-fold (3700 cpm to 13,260 cpm) in the presence of m-RNA. Under these conditions, single-chain precursors of guinea pig albumin (m.w. > 68,000) and of C4 (m.w. ≥ 205,000) were synthesized, each accounting for less than 0.5% of the newly synthesized total protein.