Use of molecular hybridization to purify and analyze albumin messenger RNA from rat liver

Abstract

A new procedure is described for purification of rat liver albumin mRNA. First, a population of RNA molecules is enriched for albumin mRNA by immunoprecipitation of polysomes containing albumin nascent chains. Poly(A)-RNA is prepared from immunoprecipitates, transcribed into complementary DNA and enriched severalfold for a particular RNA frequency component. This enriched RNA component is then purified by molecular hybridization to a limited Rot value (product of RNA concentration and incubation time), under conditions in which only the most abundant sequence component is annealed. Potentially, this procedure can be employed for the purification of a wide variety of mRNA present in lesser amounts in the cell. The isolated RNA appears to be a single frequency component by hybridization to complementary DNA transcribed from itself. This RNA is a 17S species and represents 5-8% of total cytoplasmic poly(A)-RNA. In vitro translation of the purified RNA has shown that it codes for a single polypeptide that can be identified immunoloically as albumin and migrates with rat serum albumin on sodium dodecyl sulfate/polyacrylamide gels. This albumin mRNA was determined to be essentially pure by comparing its kinetics of hybridization to those obtained with rabbit .alpha. + .beta. globin mRNA and its DNA complement. The sequence complexity of purified rat albumin mRNA corresponds to 5.9 .times. 105 daltons.