PURIFICATION OF ALBUMIN MESSENGER-RNA FROM RAT-LIVER - CONTENT OF ALBUMIN-SPECIFIC SEQUENCES IN CYTOPLASMIC AND NUCLEAR-RNA

Albumin mRNA was purified from rat liver by indirect immunoprecipitation of albumin‐synthesizing polysomes. Monospecific antibodies against albumin, raised in rabbits, were added to rat liver polysomes and the antibody‐polysome complex formed was adsorbed to goat antibodies against rabbit immunoglobulins, coupled to p‐aminobenzyl‐cellulose according to Schütz et al. [Nucleic Acid Res. 4, 71–84 (1979)]. Approximately 8–10% of the polysomes were bound to the matrix. They were then eluted from the column, RNA was isolated and mRNA recovered by chromatography on oligo(dT)‐cellulose. By translation of the mRNA in vitro in a wheat germ system, it was estimated that approximately 90% of the albumin‐synthesizing mRNA was precipitated and that the albumin mRNA fraction obtained was enriched 8–10‐fold for albumin‐coding sequences. Complementary DNA was transcribed from this mRNA. Albumin‐specific cDNA was purified by hybridization of the cDNA to polysomal rat liver mRNA up to a limited rot value of 2 × 10−2 mol · 1−1· s, isolation of the double strands by hydroxyapatite chromatography and recovery of the hybridized cDNA after alkali digestion of RNA. Kinetic data obtained by hybridization of this cDNA against total cytoplasmic and purified mRNA demonstrate that more than 90% of this cDNA comprises only one sequence component. An r0t1/2 value of 2.2 × 10−3 mol · l−1· s was estimated for the immunoprecipitated mRNA fraction. This indicates already a high degree of purity of the mRNA component of this fraction, as this RNA still contains approximately 50% ribosomal RNA. By electrophoresis of glyoxal‐denatured RNA in agarose gels, a molecular weight of 780000 was found for albumin mRNA, a value which corresponds well with a sequence complexity of 880000, estimated from the kinetics of the mRNA‐cDNA hybridization. Titrations of albumin cDNA with total polysomal poly(A)‐containing RNA indicated a content of approximately 10% for albumin mRNA in this fraction. Total nuclear RNA was shown to contain approximately 0.1% albumin‐specific sequences, whereas poly(A)‐containing nuclear RNA was about 4‐fold enriched for albumin sequences. Copyright © 1979, Wiley Blackwell. All rights reserved