Multihormonal induction of hepatic α 2u -globulin mRNA as measured by hybridization to complementary DNA

A procedure is presented for the preparation of a3 H-labeled complementary DNA (cDNA) specific for the mRNA coding for α2u -globulin, a male rat liver protein under multihormonal control that represents approximately 1% of hepatic protein synthesis. Rat liver polysomes are incubated with monospecific rabbit antiserum to α2u -globulin, which binds to the nascent α2u -globulin chains on the polysomes. These antibody-polysome complexes are then adsorbed to goat antiserum to rabbit IgG that is covalently linked top -aminobenzylcellulose. mRNA preparations are thus obtained that contain 30-40% α2u -globulin mRNA. A labeled cDNA is made to this α2u -globulin-enriched mRNA preparation by using RNA-dependent DNA polymerase (reverse transcriptase). To remove the non-α2u -globulin sequences, this cDNA preparation is hybridized to an RNA concentration × incubation time (R0 t) of 1000 mol of ribonucleotide per liter × sec with female rat liver mRNA, which, though it shares the vast majority of mRNA sequences with male liver, contains no α2u -globulin mRNA sequences. The cDNA remaining single-stranded is isolated by hydroxylapatite chromatography and is shown to be specific for α2u -globulin mRNA by several criteria.Good correlation was found in all endocrine states studied between the hepatic level of α2u -globulin, the level of functional α2u -globulin mRNA as assayed in a wheat germ cell-free translational system, and the level of α2u -globulin mRNA sequences as measured by hybridization to the α2u -globulin cDNA. Thus, the hormonal control of hepatic α2u -globulin synthesis by sex steroids and thyroid hormone occurs through modulation of the cellular level of α2u -globulin mRNA sequences, presumably by hormonal control of transcriptive synthesis.