Immunochemical isolation and characterization of ovalbumin messenger ribonucleic acid

Abstract

Hen oviduct ovalbumin messenger RNA has been purified to apparent homogeneity and its physical and molecular properties have been examined. Purification was achieved through the use of indirect immunoprecipitation to isolate ovalbumin synthesizing polysomes and the use of poly(U)-Sepharose chromatography to separate quantitatively ovalbumin messenger RNA from ribosomal RNA. Ovalbumin mRNA was purified 90 to 100-fold over oviduct polysomal RNA as judged by both the rate of hybridization to a complementary DNA and by translation in a rabbit reticulocyte lysate protein-synthesizing system. Isolated ovalbumin mRNA migrates as a single sharp symmetrical peak on sucrose gradient sedimentation and polyacrylamide gel electrophoresis. The molecular weight of ovalbumin mRNA determined by sedimentation in denaturing dimethylsulfoxide gradients is 700,000 (equivalent to 2,180 nucleotides). The complexity of purified ovalbumin mRNA determined from the relative rate of hybridization to a complementary DNA is 2,280 nucleotides. Since ovalbumin synthesis requires only 1,161 nucleotides, ovalbumin mRNA appears to contain approximately 1,150 untranslated nucleotides. The average length of the polyadenylate sequence in ovalbumin mRNA is only 44 nucleotides and it does not account for significant fraction of the untranslated nucleotides.