Application of a protein-blotting procedure to the study of human glucocorticoid receptor interactions with DNA

Journal Article · Wed Apr 01 04:00:00 EST 1987 · Proc. Natl. Acad. Sci. U.S.A.; (United States)

DOI:https://doi.org/10.1073/pnas.84.7.1744· OSTI ID:6027523

Silva, C M; Tully, D B; Petch, L A; Jewell, C M; Cidlowski, J A

To exert their effects, glucocorticoid receptor complexes interact selectively with DNA sequences known as glucocorticoid regulatory elements. The authors have studied the interaction between human glucocorticoid receptors and mouse mammary tumor virus (MMTV) DNA by means of a procedure that permits analysis after immobilization of the receptor on nitrocellulose. Proteins from crude cytosolic or nuclear extracts were electrophoresed on NaDodSO/sub 4//PAGE gels, soaked in a urea buffer to remove NaDodSO/sub 4/, transferred to nitrocellulose, and probed with nick-translated MMTV (/sup 32/P)DNA in a 5% nonfat dry milk buffer, which minimizes nonselective DNA-protein interactions. They present evidence that MMTV (/sup 32/P)DNA interacts selectively with the glucocorticoid receptor. These data include (i) comigration of (/sup 3/H)dexamethasone mesylate-labeled band and bound MMTV (/sup 32/P)DNA on gel electrophoresis systems; (ii) localization of DNA-binding activity in the cytosol of cells incubated with steroid at 0/sup 0/C and in the nucleus and cytosol of cells incubated at 37/sup 0/C; (iii) binding of the MMTV DNA to highly purified receptor; and (iv) absence of MMTV DNA binding activity in extracts from cells whose receptor has been down-regulated. Furthermore, glucocorticoid receptors analyzed under these conditions exhibit selective binding to DNA fragments that contain glucocorticoid regulatory elements.

• Research Organization: Univ. of North Carolina, Chapel Hill
• OSTI ID: 6027523
• Journal Information: Proc. Natl. Acad. Sci. U.S.A.; (United States), Journal Name: Proc. Natl. Acad. Sci. U.S.A.; (United States) Vol. 84:7; ISSN PNASA
• Country of Publication: United States
• Language: English

Related Subjects

550201* -- Biochemistry-- Tracer Techniques
59 BASIC BIOLOGICAL SCIENCES
ADRENAL HORMONES
BETA DECAY RADIOISOTOPES
BETA-MINUS DECAY RADIOISOTOPES
CONFIGURATION INTERACTION
CORTICOSTEROIDS
DAYS LIVING RADIOISOTOPES
DEXAMETHASONE
DNA
ELECTROPHORESIS
GLUCOCORTICOIDS
HELA CELLS
HYDROXY COMPOUNDS
ISOTOPE APPLICATIONS
ISOTOPES
KETONES
LABELLED COMPOUNDS
LIGHT NUCLEI
MEMBRANE PROTEINS
NUCLEI
NUCLEIC ACIDS
ODD-ODD NUCLEI
ORGANIC COMPOUNDS
PHOSPHORUS 32
PHOSPHORUS ISOTOPES
PREGNANES
PROTEINS
RADIOISOTOPES
RECEPTORS
STEROIDS
TRACER TECHNIQUES
TRITIUM COMPOUNDS