Isolation of ribonucleic acid from the unactivated rat liver glucocorticoid receptor.
We have reported that the 7-8S form of the rat liver glucocorticoid receptor is associated with RNA. Whether the unactivated 9-10S form of the glucorticoid receptor is also associated with RNA is less clear. Here we provide evidence that the unactivated 9-10S receptor is indeed associated with RNA. Unactivated 9-10S receptor was partially purified by diethylaminoethyl (DEAE)-cellulose chromatography in the presence of molybdate, an activation inhibitor. This preparation was then bound to BuGR-2, a mouse monoclonal antibody of the immunoglobulin G (IgG)-2 class to the rat liver glucocorticoid receptor, or to nonspecific mouse IgG-2. The antibody-antigen complex was then bound to protein A sepharose and washed to remove extraneous RNA. When the receptor was dissociated from the antibody and the RNA extracted and end-labeled, a distinct band of approximately 170 nucleotide (nt) was found that was specific for the BuGR-2 purified receptor. This band could also be found in DEAE-cellulose receptor that had been isolated from sucrose gradients. The DEAE-cellulose receptor was then cross-linked with formaldehyde before mixing with BuGR-2 in order to permit more vigorous washing of the antigen-antibody complex. In addition to the 170 nt RNA band, another distinct band at approximately 400 nt was seen that was specific to the BuGR-2 derived isolate. These results provide evidence that the 9-10S form of the glucocorticoid receptor from rat liver is associated with RNA.
Unger
A L
AL
Department of Biology, Carleton College, Northfield, Minnesota 55057.
Uppaluri
R
R
Ahern
S
S
Colby
J L
JL
Tymoczko
J L
JL
D000818:Animals; D000911:Antibodies, Monoclonal; D002499:Centrifugation, Density Gradient; D003432:Cross-Linking Reagents; D004591:Electrophoresis, Polyacrylamide Gel; D008099:Liver / Q000032:analysis*; D012313:RNA / Q000032:analysis / Q000302:isolation & purification*; D051381:Rats; D011965:Receptors, Glucocorticoid / Q000032:analysis* / Q000302:isolation & purification
D000911:Antibodies, Monoclonal; D003432:Cross-Linking Reagents; D011965:Receptors, Glucocorticoid; D012313:RNA