Clustered point mutation analysis of the rat prolactin promoter.
To identify DNA regions important for basal and hormone-stimulated transcription of the rat PRL gene, a series of clustered point mutations were prepared within the immediate 5' flanking region. DNA fragments representing the wild-type and 19 different linker-scanner mutations of the PRL gene were each linked to a luciferase marker gene, and the DNA constructs were transferred into GH3 pituitary tumor cells by electroporation. Luciferase activity was determined 24 h after transfection in extracts from control cells or cells treated with 0.5 mM chlorophenylthio-cAMP, 100 nM TRH, or 100 nM phorbol myristate acetate. The individual clustered point mutations covered a region from just up-stream of the TATA box (position -30) to a position 193 basepairs up-stream from the start of transcription. Five regions in which mutations produced substantial decreases in both basal and cAMP-, TRH-, or phorbol ester-stimulated expression of the marker gene were detected. Three of these regions (positions -41 to -58, -113 to -124, and -149 to -156) correspond to previously identified binding sites for the pituitary-specific, homeobox protein, Pit-1/GHF-1. The fourth and fifth regions do not correspond to Pit-1/GHF-1-binding sites and presumably represent sites for an unidentified factor. Within these regions, sequences with some similarity to a consensus cAMP response element and an AP-2-binding site have been detected. These data confirm the importance of Pit-1/GHF-1 as a key factor in PRL gene transcription. In addition, the results suggest that additional transcription factors are probably required for efficient expression of the PRL gene.(ABSTRACT TRUNCATED AT 250 WORDS)
Iverson
R A
RA
Department of Physiology and Biophysics, University of Iowa, Iowa City 52242.
Day
K H
KH
d'Emden
M
M
Day
R N
RN
Maurer
R A
RA
D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D000242:Cyclic AMP / Q000494:pharmacology; D004247:DNA / Q000235:genetics; D004268:DNA-Binding Proteins / Q000378:metabolism; D005819:Genetic Markers; D008156:Luciferases / Q000235:genetics; D008969:Molecular Sequence Data; D016296:Mutagenesis; D010911:Pituitary Neoplasms; D011388:Prolactin / Q000235:genetics*; D011401:Promoter Regions, Genetic / Q000235:genetics*; D051381:Rats; D012091:Repetitive Sequences, Nucleic Acid; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D013973:Thyrotropin-Releasing Hormone / Q000494:pharmacology; D050817:Transcription Factor Pit-1; D014157:Transcription Factors / Q000378:metabolism; D014158:Transcription, Genetic; D014162:Transfection; D014407:Tumor Cells, Cultured
D004268:DNA-Binding Proteins; D005819:Genetic Markers; C494186:Pou1f1 protein, rat; D050817:Transcription Factor Pit-1; D014157:Transcription Factors; D013973:Thyrotropin-Releasing Hormone; D011388:Prolactin; D004247:DNA; D000242:Cyclic AMP; D008156:Luciferases; D013755:Tetradecanoylphorbol Acetate