Regulation of prolactin mRNA analyzed using a specific cDNA probe.

DNA complementary to prolactin-specific sequences was prepared using a method which did not require purification of the prolactin mRNA. cDNA was synthesized using total cytoplasmic mRNA from a prolactinproducing rat pituitary cell line (GH,) as a template for avian myeloblastosis virus reverse transcriptase. A cDNA species 840 nucleotides in length was purified by electrophoresis on formamide polyacrylamide gels, eluted, and subjected to preparative hybridization using mRNA from GH, cells and to mRNA from a related cell line which does not produce prolactin (GC). The resultant cDNA hybridized specifically to mRNA from prolactin-producing cells with pseudo-first order reaction kinetics and the mRNA=cDNA hybrid exhibited a sharp melting profile with a T,,, of 88°C. The identity of the 840-nucleotide cDNA was confirmed by two-dimensional gel electrophoresis following hybridization-arrested translation. The prolactin cDNA was employed as a probe to investigate changes in prolactin-specific nucleic acid sequences during thyrotropin-releasing hormone stimulation of prolactin synthesis. Following addition of thyrotropin-releasing hormone to cultured cells, cytoplasmic prolactin mRNA sequences increased from 2,500 to 20,000 molecules/cell over 72 h, in parallel with an increase in prolactin production. In all prolactin-producing cells examined, and under a variety of hormonal conditions, the prolactin mRNA content was proportional to the rate of prolactin production. By hybridization of the prolactin cDNA to GH, DNA in cDNA excess, GH, cells were found to contain a calculated 2.4 copies of the prolactin structural gene per haploid genome.