Open AccessT-cell nuclei contain a protein that binds upstream of the murine granulocyte-macrophage colony-stimulating factor gene.
Author(s) -
Robert James,
Jan Kazenwadel
Publication year - 1989
Publication title -
proceedings of the national academy of sciences of the united states of america
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 5.011
H-Index - 771
eISSN - 1091-6490
pISSN - 0027-8424
DOI - 10.1073/pnas.86.19.7392
Subject(s) - microbiology and biotechnology , biology , gene , transcription (linguistics) , transcription factor , nuclear protein , cell culture , granulocyte macrophage colony stimulating factor , gene expression , dna , biochemistry , genetics , cytokine , philosophy , linguistics
Stimulation of a murine T-cell line (O18A) by Con A has been shown to cause a large increase in the cytoplasmic granulocyte-macrophage colony-stimulating factor (GM-CSF) mRNA level. Using run-on transcription experiments in isolated nuclei, we have shown that some of this response is from enhanced transcription of the GM-CSF gene. Changes in the transcription rate of this gene can be seen as early as 30 min after adding the Con A. With a DNA fragment mobility-shift assay on nuclear extracts from these cells we detected a protein that binds upstream of the murine GM-CSF gene. Partial purification and concentration of this protein by DEAE-Sephacel and phosphocellulose chromatography enabled us to examine its interaction with DNA in more detail. Competition and methylation interference experiments have shown that the protein binds to the sequence 3'-TCCATCAAGGGG-5' (positions -90 to -82). This sequence is contained within a region found to be involved in regulating inducible GM-CSF transcription in a human T-cell line [Miyatake, S., Seiki, M., Yoshida, M. & Arai, K. (1988) Mol. Cell. Biol. 8, 5581-5587].