Purification of Vitellogenin mRNA and Serum Albumin mRNA from Avian Liver by Preparative Gel Electrophoresis

Vitellogenin and serum albumin mRNA were isolated from liver polysomal RNA of estrogentreated hens and ducks. The vitellogenin mRNA of both avian species was purified to homogeneity in good yield by isolation of poly(A)-containing RNA and subsequent fractionation of this RNA by means of high-resolution preparative gel electrophoresis. The fractionation simultaneously yielded preparations highly enriched in serum albumin mRNA. Conditions were established to avoid the appearance of RNA aggregates and of rRNA in the final mRNA preparations. The purity of the vitellogenin mRNA from both chicken and duck was estimated to be over 95% from analysis in the electron microscope, and by gel electrophoresis and sucrose gradient centrifugation under denaturing conditions. These results were confirmed by hybridisation kinetics of mRNA with the complementary DNA. Serum albumin mRNA preparations were not completely homogeneous by these methods. Molecular weights of vitellogenin mRNA and serum albumin mRNA were determined to be (2.1 − 2.8) × 106, and (0.85 − 1.05) × 106 respectively, depending on the molecular weight standards and methods used. In an mRNA-dependent reticulocyte system, vitellogenin mRNA coded for products with a broad size range up to Mr 220000; phosphorylated and glycosylated serum vitellogenin has an apparent Mr of 240000. With serum albumin the products were more homogeneous in size, the major protein probably being pre-albumin. Immunological methods were used to identify and quantitate the proteins synthesized in vitro.