MOUSE ALBUMIN MESSENGER-RNA IN LIVER AND A HEPATOMA-CELL LINE - PREPARATION OF COMPLEMENTARY-DNA FROM PURIFIED MESSENGER-RNA AND QUANTITATION BY NUCLEIC-ACID HYBRIDIZATION

Mouse albumin mRNA was isolated by direct immunoprecipitation of albumin-synthesizing polysomes and oligo(dT)-cellulose chromatography of albumin polysomal RNA. This albumin mRNA sediments at about 17 S, corresponding to a MW of approximately 6.5 .times. 105 or 2000 nucleotides. Translation in vitro [in a wheat germ lysate] yielded a product which is immunoprecipitable with anti-mouse albumin and which showed a single radioactive peak having a MW of 68,000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. DNA, complementary to albumin mRNA, was synthesized with avian myeloblastosis virus RNA-dependent DNA polymerase. This complementary DNA was shown by alkaline sucrose density gradient sedimentation to have a MW of 5.3 .times. 105, equivalent to 1740 nucleotides and representing approximately 87% of the total 17 S mRNA. Hybridization of the c[complementary]DNA ot its template mRNA gave a Rot1/2 [half-renaturation time] value of 2.3 .times. 10-3 mol nucleotides .cntdot. S .cntdot. liter-1 (in 0.5 M NaCl). The resultant cDNA-mRNA hybrid displayed a melting temperature of 89.degree. C when analyzed by thermal elution from a hydroxylapatite column, indicating a high degree of fidelity of the base pairings formed in this hybrid. Data from the hybridization analyses and cell-free translation studies indicate that the albumin mRNA is about 80-85% pure. Quantitation of albumin mRNA in total cytoplasmic RNA by hybridization of cDNA under conditions of RNA excess revealed that mouse liver contains about 10-fold more albumin mRNA sequences than Hepa-2 cells, a permanent mouse hepatoma cell line that has maintained the capacity to synthesize albumin.