Multihormonal induction of hepatic alpha2u-globulin mRNA as measured by hybridization to complementary DNA.
A procedure is presented for the preparation of a (3)H-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 IgG 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 x incubation time (R(0)t) of 1000 mol of ribonucleotide per liter x sec 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. Thus, 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.
D000510:Alpha-Globulins / Q000235:genetics*; D000818:Animals; D000888:Antibodies, Anti-Idiotypic / Q000302:isolation & purification; D002474:Cell-Free System; D004247:DNA / Q000378:metabolism*; D006728:Hormones / Q000502:physiology*; D066298:In Vitro Techniques; D007700:Kinetics; D008099:Liver / Q000378:metabolism* / Q000648:ultrastructure; D008297:Male; D009693:Nucleic Acid Hybridization; D011132:Polyribosomes / Q000378:metabolism; D014176:Protein Biosynthesis; D012333:RNA, Messenger / Q000096:biosynthesis*; D051381:Rats
D000510:Alpha-Globulins; D000888:Antibodies, Anti-Idiotypic; D006728:Hormones; D012333:RNA, Messenger; D004247:DNA