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Isolation of Rat ₁-Fetoprotein Messenger RNA from Morris Hepatoma 7777¹

Department of Biochemistry, Vanderbilt University Medical School, Nashville, Tennessee 37232

A double-antibody procedure has been developed for the isolation of 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, migrates as a single 21S component in polyacrylamide gel electrophoresis; in a cell-free translation system, it yields a peptide product immunoprecipitable by anti-rat AFP antiserum, but not by anti-rat albumin, and which migrates slightly faster than serum AFP on sodium dodecyl sulfate-urea-polyacrylamide gels. This messenger RNA fraction was used for the synthesis of a radioactive complementary DNA. In hybridization assays, the complementary DNA reassociated with its purified template at a Cr₀t_1/2 [product of RNA concentration (mol of nucleotides per liter) x half-time (sec)] of 1.5 x 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 messenger RNA fraction, its selective translation into AFP, and the specificity of the hybridization probe indicate that the procedure described yields a highly purified rat AFP messenger RNA.

¹ Supported by USPHS Grant CA21759.

² Postdoctoral Fellow of the Medical Research Council of Canada. To whom requests for reprints should be addressed, at Laboratoire de Biologie Cellulaire a Moléculaire, Centre de Recherche de l'Hôtel-Dieu de Québec, Québec, Québec G1R 2J6, Canada.

³ Present address: Department of Biochemistry, College of Medicine, The University of Vermont, Burlington, Vt. 05401.

Received 6/20/78. Accepted 3/ 2/79.

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