Translation in Vitro of Mouse Liver mRNA Into C3, C4 and Albumin in a Protein-Synthesizing System from Rabbit Reticulocyte Lysate

In an attempt to define the molecular basis of the genetic lesions responsible for some of the inherited complement deficiency states we have initiated studies on the ability of isolated mRNA to direct the synthesis of complement components in vitro. Poly (A)-mRNA was isolated from the post-mitochondrial supernatant fraction of mouse liver homogenate by phenol-chloroform extraction and subsequent oligo (dT)-cellulose chromatography. Translation of the isolated mRNA in a reticulocyte lysate cell-free system, in the presence of ³H leucine and an energy-synthesizing system, greatly stimulated the incorporation of the labeled amino acid into protein in a time and dose-dependent fashion. Specific immunoprecipitation of the translated products with the double antibody technique showed that proteins antigenically related to C3, C4, and albumin had been synthesized. The immunoprecipitates were analyzed by SDS/polyacrylamide gel electrophoresis. The immunoprecipitated C4 radioactivity migrated predominantly in a single peak, m.w. 230,000.