Abstract
A method to localize glucocorticoid receptor (GR) by utilizing the specific interaction between GR and glucocorticoid responsive element (GRE) is described. In this study, pBR 322 DNA was thymine-thymine (T-T) dimerized by UV-irradiation and used as a probe, since pBR 322 DNA harbors GRE DNA consensus sequences and it is known that GR binds to pBR 322 DNA specifically. T-T dimerized pBR 322 DNA was incubated with fresh frozen sections of adrenalectomized rat liver, which were fixed in 4% paraformaldehyde in phosphate buffered saline, and the sites of reaction were visualized by a successive use of rabbit anti-T-T antibody and horseradish-peroxidase labeled goat anti-rabbit IgG antibody. As a result, GR was localized to the nuclei and nuclear membranes as well as cytoplasm of hepatocytes, and most of the staining was lost in the liver sections from rats, which were intraperitoneally injected with hydrocortisone. On the other hand, the staining for T-T dimerized DNA probe with cyclic AMP responsive element consensus sequence was localized solely to the nuclei and the staining pattern was not altered by an injection of the steroid, indicating that the staining for pBR 322 DNA is sequence-specific. Finally, this method should provide useful information on the localization of steroid hormone receptors with DNA-binding activity.
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