DNA and protein components of nuclear acceptor sites for androgen receptors in the rat prostate.
Androgen receptor-acceptor complexes in nuclei from rat ventral prostates were cross-linked in situ with formaldehyde and partially purified using affinity chromatography. To isolate acceptor DNA, the cross-linked receptor-acceptor complexes in formaldehyde-treated chromatin samples were adsorbed to dihydrotestosterone-17 beta-succinyl agarose, eluted with 75 microM dihydrotestosterone-1% SDS, digested with proteinase K and extracted with phenol-chloroform. After 32P end-labelling and PAGE, this DNA contained two distinct bands of DNA (about 300 and 400 base pairs respectively) which were unique relative to the total prostatic DNA. As an alternative approach for characterizing acceptor DNA, the DNA in prostatic nuclei and cross-linked chromatin was labelled with 32P by nick translation and analysed in glycerol density gradients for associations with cross-linked androgen receptors. A symmetrical 7s peak of 32P-DNA with a small amount of coincident receptor was observed in the gradients after mild trypsin treatment. In the absence of trypsin treatment, both the cross-linked receptors and the labelled DNA sedimented to the bottom of the gradients. Isolation of acceptor proteins involved iodination of cross-linked chromatin with 125I and androgen affinity chromatography. A comparison of the relative efficiency of retention and elution of 125I-proteins from different affinity columns revealed that testosterone-17 beta-succinyl agarose was potentially most suitable for purification of acceptor proteins. After electrophoresis on SDS-polyacrylamide gels, the eluates from this type of affinity matrix were found to contain two major peaks of 125I-labelled proteins--one corresponding to a protein with a similar molecular weight as the nuclear androgen receptor (33,000 Da); the other having a molecular weight of 20,000 Da. While the precise identity of this latter entity is unknown, its enrichment and retention by the affinity gel implies that it is closely associated with the androgen receptor and may be a component of the acceptor sites.
Department of Cancer Endocrinology, Cancer Control Agency of British Columbia, Vancouver, Canada.
P S
PS
Rennie
J F
JF
Bowden
N
N
Bruchovsky
P S
PS
Frenette
J A
JA
Foekens
H
H
Cheng
D000818:Animals; D002467:Cell Nucleus / Q000378:metabolism*; D002843:Chromatin / Q000032:analysis / Q000187:drug effects; D002846:Chromatography, Affinity; D002868:Chromosomal Proteins, Non-Histone / Q000302:isolation & purification / Q000378:metabolism*; D003432:Cross-Linking Reagents / Q000494:pharmacology; D004247:DNA / Q000302:isolation & purification / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000378:metabolism; D013196:Dihydrotestosterone / Q000378:metabolism; D008297:Male; D011467:Prostate / Q000378:metabolism*; D051381:Rats; D011919:Rats, Inbred Strains; D011944:Receptors, Androgen / Q000187:drug effects / Q000378:metabolism*
D002843:Chromatin; D002868:Chromosomal Proteins, Non-Histone; D003432:Cross-Linking Reagents; D004268:DNA-Binding Proteins; D011944:Receptors, Androgen; D013196:Dihydrotestosterone; D004247:DNA