Immunochemical isolation of prolactin messenger RNA

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12 Scopus citations

Abstract

Prolactin mRNA has been isolated using immunochemical techniques. Initial experiments demonstrated that 125I-labeled prolactin antibody was able to bind to pituitary polysomes but not to liver polysomes, suggesting that the binding is specific. Prolactin-synthesizing polysomes were immunoprecipitated by reaction with antiprolactin followed by anti-antibody. Immunoprecipitated polysomal RNA was chromatographed on oligo(dT)-cellulose, and the poly(A) RNA was sedimented through a sucrose gradient. This procedure resulted in a 32-fold purification of prolactin mRNA as determined by translation in a mRNA-dependent reticulocyte lysate assay. Translation analysis also suggested that the isolated prolactin mRNA is greater than 95% pure. The molecular weight of prolactin mRNA determined by electrophoresis on agarose gels containing 10 mM methyl mercury hydroxide was 350,000. Purified prolactin mRNA was used to synthesize full-length cDNA by means of avian myeloblastosis reverse transcriptase. Use of this cDNA as a hydbridization probe demonstrated that estrogen is able to increase the concentration of prolactin mRNA sequences in the pituitary.

Original languageEnglish (US)
Pages (from-to)854-859
Number of pages6
JournalJournal of Biological Chemistry
Volume255
Issue number3
StatePublished - Jan 1 1980

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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