Characterization of human glucocorticoid receptor complexes formed with DNA fragments containing or lacking glucocorticoid response elements.
Sucrose density gradient shift assays were used to study the interactions of human glucocorticoid receptors (GR) with small DNA fragments either containing or lacking glucocorticoid response element (GRE) DNA consensus sequences. When crude cytoplasmic extracts containing [3H]triamcinolone acetonide [( 3H]TA) labeled GR were incubated with unlabeled DNA under conditions of DNA excess, a GRE-containing DNA fragment obtained from the 5' long terminal repeat of mouse mammary tumor virus (MMTV LTR) formed a stable 12-16S complex with activated, but not nonactivated, [3H]TA receptor. By contrast, if the cytosols were treated with calf thymus DNA-cellulose to deplete non-GR-DNA-binding proteins prior to heat activation, a smaller 7-10S complex was formed with the MMTV LTR DNA fragment. When similar experiments were conducted under conditions of large receptor excess, using 3' [32P]-MMTV LTR DNA, the trace quantity of DNA formed a stable 10-14S complex with DNA-cellulose pretreated cytosols or with untreated cytosols in the presence of excess Escherichia coli competitor DNA. If trace quantities of the 3' [32P]-MMTV LTR DNA were incubated with untreated crude cytosols, much larger complexes were formed, indicating the association of other cytosolic proteins with the MMTV LTR DNA fragment. Activated [3H]TA receptor from DNA-cellulose pretreated cytosols also interacted with two similarly sized fragments from pBR322 DNA, but with lower apparent affinities in the order MMTV LTR DNA fragment much greater than pBR322 fragment containing a single GRE DNA consensus sequence greater than non-GRE-containing pBR322 fragment.(ABSTRACT TRUNCATED AT 250 WORDS)
Tully
D B
DB
Department of Biochemistry, University of North Carolina, Chapel Hill 27599-7545.
Cidlowski
J A
JA
D001483:Base Sequence; D002460:Cell Line; D002482:Cellulose / Q000031:analogs & derivatives; D002499:Centrifugation, Density Gradient; D004247:DNA / Q000031:analogs & derivatives / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000302:isolation & purification / Q000378:metabolism; D005786:Gene Expression Regulation; D006801:Humans; D006863:Hydrogen-Ion Concentration; D010957:Plasmids; D011965:Receptors, Glucocorticoid / Q000378:metabolism*; D012492:Salts
D004268:DNA-Binding Proteins; C033190:DNA-cellulose; D011965:Receptors, Glucocorticoid; D012492:Salts; D002482:Cellulose; D004247:DNA