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

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Abstract
A procedure is presented for the preparation of a 3H-labeled complementary DNA (cDNA) specific for the mRNA coding for .alpha.2u-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 .alpha.2u-globulin, which binds to the nascent .alpha.2u-globulin chains on the polysomes. These antibody-polysome complexes are then adsorbed to goat antiserum to rabbit Ig[immunoglobulin]G that is covalently linked to p-aminobenzylcellulose. mRNA preparations are thus obtained that contain 30-40% .alpha.2u-globulin mRNA. A labeled cDNA is made to this .alpha.2u-globulin-enriched mRNA preparation by using RNA-dependent DNA polymerase (reverse transcriptase). To remove the non-.alpha.2u-globulin sequences, this cDNA preparation is hybridized to an RNA concentration .times. incubation time (R0t) of 1000 mol of ribonucleotide/l .times. s with female rat liver mRNA, which, though it shares the vast majority of mRNA sequences with male liver, contains no .alpha.2u-globulin mRNA sequences. The cDNA remaining single-stranded is isolated by hydroxylapatite chromatography and is shown to be specific for .alpha.2u-globulin mRNA by several criteria. Good correlation was found in all endocrine states studied between the hepatic level of .alpha.2u-globulin, the level of functional .alpha.2u-globulin mRNA as assayed in a wheat germ cell-free translational system, and the level of .alpha.2u-globulin mRNA sequences as measured by hybridization to the .alpha.2u-globulin cDNA. The hormonal control of hepatic .alpha.2u-globulin synthesis by sex steroids and thyroid hormone occurs through modulation of the cellular level of .alpha.2u-globulin mRNA sequences, presumably by hormonal control of transcriptive synthesis.