Identification, cloning and analysis of prolactin-regulated genes in the pigeon crop

My objective was to investigate the molecular actions of prolactin in the pigeon crop system. The pigeon crop, upon stimulation by prolactin, proliferates extensively and undergoes a simple differentiative process. I have identified specific crop gene products regulated by prolactin and analyzed parameters of their gene expression. Identification of gene products whose expression was regulated by prolactin was done in two ways. In vivo labelling identified peptides of 35,000 MW, 92,000 MW, and 58,000 MW (lipid-soluble) as prolactin inducible. Secondly, induction at the level of mRNA was examined using cell-free translation. Prolactin greatly repressed crop mRNAs coding for 16,000 and 22,000 MW peptides and induced the accumulation of a mRNA coding for a 25,000 MW protein. The direct action of prolactin on the crop was shown by using local injection of prolactin in one lobe of the crop and injecting saline solution into the contralateral lobe. CP25 was detected only in the prolactin injected side. Recombinant DNA techniques were used to construct and screen a pigeon crop cDNA library. Prolactin-induced crop mRNA was used as a template for reverse transcriptase production of cDNA. The cDNA was made double stranded, C-tailed, inserted into the G-tailed Pst 1 site of pBR322, and transformed into E. coli HB101. A screen of the library with cDNA produced from both prolactin-induced and non-induced crop mRNA showed that about two percent of the clones preferentially hybridized to prolactin-induced sequences. Northern analysis revealed one clone (DA4) to be complementary to an mRNA species of 1400 bases that is generally undetectable in naive crop and abundant in hormonally stimulated tissue. Hybrid selection showed DA4mRNA coded for a peptide of 35,000 MW. The dose response for DA4mRNA induction by prolactin was a biphasic curve with the greatest response at a 200 ug PRL/day dose. Using this dose, DA4mRNA accumulation was detectable at three hours after a single injection of prolactin and continued to accumulate through 24 hours. Hormones with known (oPRL, hGH, hPL) and suspected (bPL) lactogenic activities promoted DA4mRNA accumulation, while nonlactogenic hormones (bGH, insulin) were inactive. Inhibitor experiments showed DA4mRNA accumulation is dependent on ongoing protein synthesis. These studies have examined prolactin\u27s actions on pigeon crop at the molecular level and have shown prolactin acts to induce and repress the expression of specific gene products in the crop