Enrichment of a Second Class of Native Acceptor Sites for the Avian Oviduct Progesterone Receptor as Intact Chromatin Fragments

Michael Horton, James P. Landers, Malayannan Subramaniam, Barb Gosse, Thomas C. Spelsberg, Amy Goldberger, Hiroo Toyoda

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Abstract

Several classes of specific progesterone receptor (PR) nuclear binding sites (acceptor sites) have previously been identified in avian oviduct chromatin on the basis of different binding affinities. Recently, two classes of acceptor proteins (AP) that are associated with these binding sites in the avian oviduct have been identified. These APs were termed receptor binding factors (RBF-1 and -2), and one (RBF-1) has been purified [Schuchard et al. (1991) Biochemistry 30, 4535-4542]. The RBF-1 is associated with the highest affinity class of sites in the intact chromatin, and the RBF-2 is associated with the second highest affinity class of sites. The PR binding sites and their associated RBF-2 protein remain with the residual chromatin fraction following extraction by 4 M Gdn-HCl. This Gdn-HCl-treated chromatin has been termed nucleoacidic protein (NAP). This paper describes the 200-fold enrichment of the native RBF-2 class of PR acceptor sites beginning with the DNase I digestion of NAP to obtain DNase-resistant fragment (NAPf) containing ~ 150 bp of DNA. The PR binding sites are further enriched by high-performance or fast protein liquid chromatography and chromatofocusing. Anti-RBF-1/RBF-2 protein antibodies identify antigens that coelute with the PR binding activity. Hybridization analysis of the DNAf from the enriched NAPf demonstrates sequence homologies with the nuclear matrix DNA as well as with genomic sequences of the rapid steroid responding nuclear protooncogenes c-myc and c-jun. However, comparative analyses of the whole genomic DNA with the nuclear matrix DNA indicate that the RBF-2 (NAPf) is largely nonnuclear matrix. These data show that discrete chromatin fragments containing the native nuclear acceptor sites for the avian oviduct PR can be isolated and used to analyze the structure and composition of these sites.

Original languageEnglish (US)
Pages (from-to)9523-9530
Number of pages8
JournalBiochemistry
Volume30
Issue number39
DOIs
StatePublished - Oct 1 1991

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ASJC Scopus subject areas

  • Biochemistry

Cite this

Horton, M., Landers, J. P., Subramaniam, M., Gosse, B., Spelsberg, T. C., Goldberger, A., & Toyoda, H. (1991). Enrichment of a Second Class of Native Acceptor Sites for the Avian Oviduct Progesterone Receptor as Intact Chromatin Fragments. Biochemistry, 30(39), 9523-9530. https://doi.org/10.1021/bi00103a020