Clustered point mutation analysis of the rat prolactin promoter

Ronald A. Iverson, Kathleen H. Day, Michael D'Emden, Richard Day, Richard A. Maurer

Research output: Contribution to journalArticle

102 Citations (Scopus)

Abstract

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 Pit1/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. Several of the linker-scanner mutations appeared to reduce the ability of cAMP and phorbol esters to stimulate gene expression, including mutations in Pit-1-binding sites as well as the putative cAMP response element and AP-2-binding site. These findings suggest a role for Pit-1 as well as other transcription factors in mediating both basal and multihormone-stimulated PRL gene transcription.

Original languageEnglish (US)
Pages (from-to)1564-1571
Number of pages8
JournalMolecular Endocrinology
Volume4
Issue number10
StatePublished - 1990
Externally publishedYes

Fingerprint

Point Mutation
Prolactin
Binding Sites
Mutation
Genes
Response Elements
Phorbol Esters
Luciferases
Gene Expression
Transcription Factors
Homeodomain Proteins
TATA Box
Electroporation
5' Flanking Region
DNA
Pituitary Neoplasms
Tetradecanoylphorbol Acetate
Cell Extracts
Genetic Markers
Transfection

ASJC Scopus subject areas

  • Molecular Biology
  • Endocrinology, Diabetes and Metabolism

Cite this

Iverson, R. A., Day, K. H., D'Emden, M., Day, R., & Maurer, R. A. (1990). Clustered point mutation analysis of the rat prolactin promoter. Molecular Endocrinology, 4(10), 1564-1571.

Clustered point mutation analysis of the rat prolactin promoter. / Iverson, Ronald A.; Day, Kathleen H.; D'Emden, Michael; Day, Richard; Maurer, Richard A.

In: Molecular Endocrinology, Vol. 4, No. 10, 1990, p. 1564-1571.

Research output: Contribution to journalArticle

Iverson, RA, Day, KH, D'Emden, M, Day, R & Maurer, RA 1990, 'Clustered point mutation analysis of the rat prolactin promoter', Molecular Endocrinology, vol. 4, no. 10, pp. 1564-1571.
Iverson RA, Day KH, D'Emden M, Day R, Maurer RA. Clustered point mutation analysis of the rat prolactin promoter. Molecular Endocrinology. 1990;4(10):1564-1571.
Iverson, Ronald A. ; Day, Kathleen H. ; D'Emden, Michael ; Day, Richard ; Maurer, Richard A. / Clustered point mutation analysis of the rat prolactin promoter. In: Molecular Endocrinology. 1990 ; Vol. 4, No. 10. pp. 1564-1571.
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