Reconstitution of cell-type-specific transcription of the rat prolactin gene in vitro.
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.
Cao
Z D
ZD
Department of Cellular & Structural Biology, University of Texas Health Science Center at San Antonio 78284.
Barron
E A
EA
Carillo
A J
AJ
Sharp
Z D
ZD
D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D002467:Cell Nucleus / Q000502:physiology; D002474:Cell-Free System; D004278:DNA, Superhelical / Q000502:physiology; D004268:DNA-Binding Proteins / Q000502:physiology; D005786:Gene Expression Regulation; D008969:Molecular Sequence Data; D010902:Pituitary Gland / Q000502:physiology; D011388:Prolactin / Q000235:genetics*; D051381:Rats; D012045:Regulatory Sequences, Nucleic Acid; D013698:Templates, Genetic; D014157:Transcription Factors / Q000502:physiology*; D014158:Transcription, Genetic
D004278:DNA, Superhelical; D004268:DNA-Binding Proteins; D014157:Transcription Factors; D011388:Prolactin