ISOLATION OF RAT ALPHA-1-FETOPROTEIN MESSENGER-RNA FROM MORRIS-HEPATOMA-7777

Abstract

A double-antibody procedure was developed for the isolation of .alpha.1-fetoprotein (AFP)-synthesizing polysomes from Morris hepatoma 7777. The polyadenylic acid-containing RNA, subsequently purified by differential sedimentation on sucrose gradient and oligodeoxythymidylic acid-cellulose chromatography, migrated as a single 21S component in polyacrylamide gel electrophoresis. In a cell-free translation system, it yielded a peptide product immunoprecipitable by anti-rat AFP antiserum, but not by anti-rat albumin, and which migrated slightly faster than serum AFP on sodium dodecyl sulfate-urea-polyacrylamide gels. This mRNA fraction was used for the synthesis of a radioactive complementary DNA. In hybridization assays, the complementary DNA reassociated with its purified template at a Crot1/2 [product of RNA concentration (mol of nucleotides/l) .times. half-time (s)] of 1.5 .times. 10-2. By reference to this value, hybridizable sequences were found to constitute 3, 2 and < 0.01% of total polyadenylic acid-containing polysomal RNA of Morris hepatoma 7777, 10 day old rat liver and adult rat liver, respectively. The high specificity of the polysome immunoprecipitation system, the electrophoretic homogeneity of the isolated mRNA fraction, its selective translation into AFP and the specificity of the hybridization probe indicated that this procedure yields a highly purified rat AFP mRNA.