Mouse albumin mRNA in liver and a hepatoma cell line. Preparation of complementary DNA from purified mRNA and quantitation by nucleic acid hybridization

P. C. Brown, J. Papaconstantinou

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Abstract

Albumin, a major serum protein synthesized and secreted by the liver, is one of several serum proteins whose synthesis is regulated by hormonal and nitritional factors as well as during liver development. As a part of our studies on the regulation of serum protein synthesis, we have isolated mouse albumin mRNA by direct immunoprecipitation of albumin-synthesizing polysomes and oligo(dT)-cellulose chromatography of albumin polysomal RNA. This albumin mRNA sediments at about 17 S, which corresponds to a molecular weight of approximately 6.5 x 105 or 2,000 nucleotides. Translation in vitro yielded a product which is immunoprecipitable with anti-mouse albumin and which showed a single radioactive peak having a molecular weight of 68,000 on sodium dedecyl 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 molecular weight of 5.3 x 105 which is equivalent to 1,740 nucleotides and represents approximately 87% of the total 17 S mRNA. Hybridization of the cDNA to its template mRNA gave a Rot(1/2) value of 2.3 x 10-3 mol nucleotides.s.liter-1 (in 0.5 M NaC1). The resultant cDNA-mRNA hybrid displayed a melting temperature of 89°C when analyzed by thermal elution from a hydroxilapatite 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 to 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.

Original languageEnglish (US)
Pages (from-to)5177-5183
Number of pages7
JournalJournal of Biological Chemistry
Volume254
Issue number12
StatePublished - Dec 1 1979
Externally publishedYes

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ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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