Albumin messenger RNA in mouse liver and a mouse hepatoma cell line

The subject of this dissertation concerns the isolation and characterization of mouse liver albumin messenger RNA (mRNA), the synthesis of complementary DNA (cDNA), and the use of this cDNA in the analysis of albumin synthesis in a cell line derived from a mouse hepatoma.

Albumin mRNA was purified from mouse liver by specific immunoprecipitation of albumin synthesizing polysomes. Initial investigations, employing ¹²⁵. I-labeled anti-mouse serum albumin, revealed that these antibodies specifically bind to albumin-synthesizing polysomes by immunologically recognizing nascent albumin polypeptides. Albumin mRNA, isolated by immunoprecipitation and selection by oligo (dT)-cellulose chromatography, was translated in a cell-free system derived from wheat gern and was shown to be enriched approximately l0-12 fold when compared to total liver polysomal RNA. This poly(A)-containing albumin mRNA exhibited a sedimentation coefficient of approximately 17S, as judged by sedimentation in aqueous sucrose gradients, and the purity of the isolated species was in excess of 80%.

Approximately full length cDNA to albumin mRNA was synthesized using avian myeloblastosis virus reverse transcriptase. This cDNA probe, when reassociated with template albumin mRNA, displayed the kinetics expected of a molecule of about 80% purity, Rot _½=2x10³ mol·sec· 1^-1Employing this probe in RNA excess hybridizations to mouse liver and to hepatoma cell cytoplasmic RNA, albumin mRNA levels in liver were approximately l0-fold higher than albumin mRNA levels in hepatoma cells.

The effects of hydrocortisone and of analogs of cyclic AMp on albumin synthesis in hepatoma cells was investigated. Albumin synthesis was increased about 2.5 and 3 fold by hydrocortisone and cyclic AMP, respectively, after 72 hours of treatment with each compound. Combined administration of these drugs resulted in a l0-fold increase in albumin synthesis, an increase that was greater than additive for the two compounds given separately. Corresponding increases in albumin mRNA levels, as measured by RNA excess hybridizations to albumin cDNA, were in complete agreement with increases in albumin synthesis, suggesting that both hydrocortisone and cyclic AMP act to increase translatable albumin mRNA in hepatoma cells and therefore may be important regulators of albumin synthesis in vivo.