Immunochemical isolation of prolactin messenger RNA.
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 320-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 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 hybridization probe demonstrated that estrogen is able to increase the concentration of prolactin mRNA sequences in the pituitary.
D000818:Animals; D005260:Female; D008970:Molecular Weight; D009693:Nucleic Acid Hybridization; D010903:Pituitary Gland, Anterior / Q000378:metabolism*; D011132:Polyribosomes / Q000378:metabolism; D011388:Prolactin / Q000096:biosynthesis*; D014176:Protein Biosynthesis; D012333:RNA, Messenger / Q000302:isolation & purification* / Q000378:metabolism; D051381:Rats
D012333:RNA, Messenger; D011388:Prolactin