Reconstitution of cell-type-specific transcription of the rat prolactin gene in vitro.

Z. D. Cao, E. A. Barron, A. J. Carillo, Z. D. Sharp

Research output: Contribution to journalArticlepeer-review

33 Scopus citations

Abstract

We present evidence for the existence of prolactin upstream factor 1 (PUF-1) in rat pituitary-derived cells and demonstrate its interaction with a symmetrical DNA element located in the 5' flanking region of the gene. An in vitro expression system developed from pituitary-derived GH3 cells was used to determine that 420 base pairs (bp) of 5' flanking DNA was sufficient for cell-specific, accurate, and efficient RNA polymerase II transcription of the rat prolactin gene. Reconstitution of in vitro transcription with pituitary and nonpituitary nuclear extracts suggested that the presence of GH3 cell-specific factors mediated the activation of prolactin gene expression. We also demonstrated that a functionally stable transcription complex assembled on the prolactin promoter. Using DNase I protection procedures, we have identified the DNA-protein binding area in the prolactin 5' flanking region. GH3 nuclear extracts contain a cell-specific protein (PUF-I) that binds to a 28-bp region (-63 to -36)which contains an 18-bp imperfect palindrome (-63 to -46). The role that the interaction between PUF-I and the imperfect palindrome plays in in vitro pituitary-specific prolactin gene expression is discussed.

Original languageEnglish (US)
Pages (from-to)3402-3408
Number of pages7
JournalMolecular and cellular biology
Volume7
Issue number10
DOIs
StatePublished - Oct 1987

ASJC Scopus subject areas

  • Molecular Biology
  • Cell Biology

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