Partial purification and preparation of polyclonal antibodies against candidate chromatin acceptor proteins for the avian oviduct progesterone receptor

Goldberger, Amy; Spelsberg, Thomas C.

doi:10.1021/bi00406a043

Cited by 11 publications

(11 citation statements)

References 33 publications

(59 reference statements)

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“…Pooled fraction 3 from the CL-Sepharose-6B chromatography was applied to preparative SDS-PAGE by use of a slab gel apparatus as described in Materials and Methods. The purified protein, isolated by this method, has the same amino acid sequence and PR acceptor activity as RBF-1, which was isolated by the longer method of Goldberger and Spelsberg (1988). The protein yield was increased 150-fold, and significant reduction in effort was achieved with this new approach.…”

Section: Resultsmentioning

confidence: 99%

Characterization of a purified chromatin acceptor protein (receptor binding factor 1) for the avian oviduct progesterone receptor

Schuchard¹,

Rejman²,

McCormick³

et al. 1991

Biochemistry

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The specific, high-affinity binding of the avian oviduct progesterone receptor (PR) with target-cell nuclei and chromatin has been shown to involve DNA complexed with specific chromatin acceptor proteins. One of these chromatin acceptor proteins has been partially purified and found to be a small hydrophobic protein with a broad pI of 5.0-6.0 [Goldberger, A., & Spelsberg, T. C., (1988) Biochemistry 27, 2103-2109]. This paper describes the final purification over 100,000-fold to apparent homogeneity of this candidate PR acceptor protein, termed the receptor binding factor 1 (RBF-1). When the avian genomic DNA is bound by RBF-1, saturable, high-affinity (KD approximately 2 x 10(-9) M) binding sites for PR are generated. RBF-1 has a unique, hydrophobic N-terminal sequence. The PR binding to the RBF-1-DNA complexes is shown to be dependent on an intact activated PR with which excess nonradiolabeled PR can compete. By use of a new, highly specific monoclonal antibody (mAb) to the RBF-1 with Western immunoblotting, RBF-1 was shown to be localized in the nucleus and to be tissue and species specific. Selective removal of the chromatin proteins containing RBF-1 results in the loss of the highest affinity class of PR binding sites. A second class of residual PR binding sites remains in the nucleoacidic protein (NAP), a complex of proteins more tightly bound to the DNA. This class of PR binding activity has been classified as the RBF-2. The RBF-1 is estimated to be 0.03% of the total chromatin protein with about 1.2 x 10(5) molecules/diploid cell.

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Section: Resultsmentioning

confidence: 99%

Characterization of a purified chromatin acceptor protein (receptor binding factor 1) for the avian oviduct progesterone receptor

Schuchard¹,

Rejman²,

McCormick³

et al. 1991

Biochemistry

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“…RBF-1 has an apparent molecular weight of 10K, a unique N-terminal sequence, and an acidic p/ and is hydrophobic. When bound to hen genomic DNA, this protein generates high-affinity, saturable binding of isolated oviduct [3H]PR to the RBF-l-DNA duplex (Goldberger et al, 1988; Schuchard et al, 1991). This protein can be dissociated from nuclei or chromatin by 4 M Gdn-HCl or by 0.2% (v/v) Triton X-100.…”

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confidence: 99%

Nuclear matrix localization and specific matrix DNA binding by receptor binding factor 1 of the avian oviduct progesterone receptor

et al. 1991

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A chromatin acceptor protein for the avian oviduct progesterone receptor (PR), termed receptor binding factor 1 (RBF-1), has recently been shown to (1) be a component of the nuclear binding sites (acceptor sites) for PR and (2) generate high-affinity binding sites (termed the RBF-1 class of sites) on avian genomic DNA [Schuchard et al. (1991) Biochemistry 30, 4535-4542]. A second class of sites and its associated protein (termed RBF-2) were also identified. This paper demonstrates that RBF-1 and also the PR nuclear binding sites are localized in the oviduct nuclear matrix. RBF-1 is found in abundance in the nuclear matrix of liver but only in traces in the nuclear matrix of spleen. Extraction of the nuclear matrix with 4.0 M Gdn-HCl results in the complete removal of RBF-1 as occurs with whole chromatin. Interestingly, a second class of specific PR binding, termed RBF-2, remains on the nuclear matrix after the removal of all RBF-1. Southern blot analysis indicates that the nuclear matrix DNA contains sequences homologous with the 5'-flanking domains of the rapidly steroid regulated c-myc and c-jun protooncogenes and the beta-actin gene, but not genomic sequences of the late sex steroid regulated gene, ovalbumin, or the alpha-actin gene. A specific, small region in the 5'-flanking domain of the c-myc gene appears to be associated with the nuclear matrix. Southwestern blot analysis using partially purified RBF-1 shows a marked affinity and specificity of the RBF-1 for the nuclear matrix DNA.(ABSTRACT TRUNCATED AT 250 WORDS)

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“…Chromosomal proteins were size-fractionated by molecular sieve chromatography as described previously (46). Fractions containing proteins in the 4 -20-kDa size range were pooled and separated by preparative SDS-polyacrylamide gel electrophoresis in a Tris-Tricine buffer as described previously (28).…”

Section: Isolation and Purification Of Rbf By Preparativementioning

confidence: 99%

“…The gel was transferred to six sheets of polyvinylidene difluoride membrane using a CAPS buffer system (10 mM CAPS ϩ 10% methanol, pH 11.0). The sixth sheet was immunostained for RBF utilizing a RBF-specific monoclonal antibody (22,46,47). The five additional sheets were matched against the one with the visible immunoproduct.…”

Section: Isolation and Purification Of Rbf By Preparativementioning

confidence: 99%

A DNA-binding Element for a Steroid Receptor-binding Factor Is Flanked by Dual Nuclear Matrix DNA Attachment Sites in the c-myc Gene Promoter

Lauber

Barrett

Subramaniam

et al. 1997

Journal of Biological Chemistry

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The receptor-binding factor (RBF) for the avian oviduct progesterone (Pg) receptor (PR) has previously been shown to be a unique 10-kDa nuclear matrix protein that generates high affinity PR-binding sites on avian DNA. This paper describes the use of Southwestern blot and DNA gel shift analyses with RBF protein to identify a minimal 54-base pair RBF-binding element in the matrix-associated region (MAR) of the Pg-regulated c-myc gene promoter. This element contains a 5 -GC-rich domain and a 3 -AT-rich domain, the latter of which has a homopurine/homopyrimidine structure. The gel shift assays required the generation of an RBF-maltose fusion protein (RBF-MBP), which specifically binds this element and is supershifted when the anti-RBF polyclonal antibody is added. Computer analysis of the fulllength amino acid sequence for RBF predicts a DNAbinding motif involving a ␤-sheet structure at the N-terminal domain. Southern blot analyses using nuclear matrix DNA suggests that there are dual MAR sites in the c-myc promoter, which flank an intervening domain containing the RBF element. The co-transfection of this MAR sequence, containing the RBF element and cloned into a luciferase reporter vector, together with an RBF expression vector construct, into steroid treated human MCF-7 cells, results in a decrease of the c-myc promoter activity relative to control transfections containing only the parent vector of the RBF expression construct. These data suggest that a unique chromatin/ nuclear matrix structure, composed of the RBF-DNA element complex which is flanked by nuclear matrix attachment sites, serves to bind the PR and repress the c-myc promoter.The classic model of steroid hormone action suggests that ligand-bound receptors dimerize and bind to specific sites (acceptor sites) on the chromatin/DNA to alter gene expression. In many genes, the steroid receptors (SR) 1 bind to specific cisacting DNA sequences, referred to as hormone response elements (for reviews, see Refs. 1-3). These elements function as enhancer-like DNA sequences and are required for the steroid hormone regulation of transcription. However, not all steroidresponsive genes contain these elements, and several other pathways and participating nuclear proteins have now been identified. The SR regulation of gene transcription has now been shown to involve 1) interactions of the SR with transcription factors, such as the AP-1 complex (for review, see Refs. 2-4); 2) interactions of SR with accessory factors, which appear to function by assisting the binding of the receptor to the steroid response elements, or to transcription factors (for reviews see Refs. 4 -6 and references therein); and finally, 3) interactions of SR with specific chromatin structures including the nuclear matrix (see Refs. 7-15 and references therein). In the latter, a class of high affinity binding sites (acceptor sites) for SRs in the nuclei, chromatin, and nuclear matrix DNAbinding proteins (e.g. acceptor proteins) has been reported by many laboratories for most of the steroid hormone...

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Partial purification and preparation of polyclonal antibodies against candidate chromatin acceptor proteins for the avian oviduct progesterone receptor

Cited by 11 publications

References 33 publications

Characterization of a purified chromatin acceptor protein (receptor binding factor 1) for the avian oviduct progesterone receptor

Characterization of a purified chromatin acceptor protein (receptor binding factor 1) for the avian oviduct progesterone receptor

Nuclear matrix localization and specific matrix DNA binding by receptor binding factor 1 of the avian oviduct progesterone receptor

A DNA-binding Element for a Steroid Receptor-binding Factor Is Flanked by Dual Nuclear Matrix DNA Attachment Sites in the c-myc Gene Promoter

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