title: Defective distal regulatory element at the 5' upstream of rat prolactin gene of steroid-nonresponsive GH-subclone.
abstract: The prolactin-nonproducing (PRL-) GH cell strains (rat pituitary tumor cells in culture). GH12C1 and F1BGH12C1, do not respond to steroid hormones estradiol or hydrocortisone (HC). However, the stimulatory effect of estradiol and the inhibitory effect of hydrocortisone on prolactin synthesis can be demonstrated in the prolactin-producing GH cell strain, GH4C1. In this investigation we have examined the 5' end flanking region of rat prolactin (rat PRL) gene of steroid-responsive, GH4C1 cells to identify the positive and negative regulatory elements and to verify the status of these elements in steroid-nonresponsive F1BGH12C1 cells. Results presented in this report demonstrate that the basel level expression of the co-transferred Neo gene (neomycin phosphoribosyl transferase) is modulated by the distal upstream regulatory elements of rat PRL gene in response to steroid hormones. The expression of adjacent Neo gene is inhibited by dexamethasone and is stimulated by estradiol in transfectants carrying distal regulatory elements (SRE) of steroid-responsive cells. These responses are not observed in transfectants with the rat PRL upstream sequences derived from steroid-nonresponsive cells. The basal level expression of the host cell alpha-2 tubulin gene is not affected by dexamethasone. We report here the identification of the distal steroid regulatory element (SRE) located between 3.8 and 7.8 kb upstream of the transcription initiation site of rat PRL gene. Both the positive and the negative effects of steroid hormones can be identified within this upstream sequence. This distal SRE appears to be nonfunctional in steroid-nonresponsive cells. Though the proximal SRE is functional, the defect in the distal SRE makes the GH substrain nonresponsive to steroid hormones. These results suggest that both the proximal and the distal SREs are essential for the mediation of action of steroid hormones in GH cells.
mesh_terms: D000818:Animals; D001426:Bacterial Proteins / Q000096:biosynthesis; D004958:Estradiol / Q000494:pharmacology; D005786:Gene Expression Regulation / Q000187:drug effects; D006854:Hydrocortisone / Q000494:pharmacology; D019868:Kanamycin Kinase; D010770:Phosphotransferases / Q000096:biosynthesis; D010911:Pituitary Neoplasms / Q000473:pathology*; D011061:Poly A / Q000096:biosynthesis; D011388:Prolactin / Q000235:genetics*; D012333:RNA, Messenger / Q000096:biosynthesis; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis; D012045:Regulatory Sequences, Nucleic Acid; D014407:Tumor Cells, Cultured / Q000032:analysis* / Q000187:drug effects
chemical_list: D001426:Bacterial Proteins; D012333:RNA, Messenger; D011993:Recombinant Fusion Proteins; D011061:Poly A; D004958:Estradiol; D011388:Prolactin; D010770:Phosphotransferases; D019868:Kanamycin Kinase; D006854:Hydrocortisone