Isolation of Rat Prolactin Messenger Ribonucleic Acid and Synthesis of the Complementary Deoxyribonucleic Acid*

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Abstract
PRL mRNA was purified from rat pituitaries. To reduce GH mRNA content and to enrich the fraction of PRL mRNA, rats were thyroidectomized and treated with estradiol. PRL mRNA was purified from the total RNA extract by poly(U)-Sepharose chromatography, followed by sucrose gradient centrifugation. The final PRL mRNA preparation sedimented with a size of approximately US on sucrose gradient centrifugation. The PRL mRNA preparation was 81% pure, and although GH mRNA has a similar sedimentation coefficient, it represented only 2.1% of the total mRNA activity as assessed by cell-free translation using nuclease-treated reticulocyte lysate. Purified PRL mRNA was used as a template for the synthesis of complementary DNA (cDNA) by reverse transcriptase. Estimation of the size of the cDNA by electrophoresis in alkaline agarose gel revealed a major 880 nucleotide species, large enough to contain most of the information present in PRL mRNA. Back hybridization of the cDNA to the mRNA template showed a saturation of 80% a nd Rot,/2 of 5.6 × 10” M × s. [Rot, the roduct of RNA concentration (molarity of nucleotides) and time (seconds) of the hybridization reaction, is measured in M s; Roti/2 = Rot for half-completion of the hybridization reaction.] No significant cross-hybridization could be demonstrated between PRL cDNA and GH mRNA purified from a transplantable pituitary tumor. After estradiol administration to intact male rats, elevation of PRL mRNA levels, as determined by hybridization, correlated well with that assessed by translation.