Biomedical subjects

F V Peale

Publications and source records attributed to F V Peale.

5 recordsLinked to original sources

PubMed1992

Cooperative estrogen receptor interaction with consensus or variant estrogen responsive elements in vitro.

Specific binding of estradiol-liganded, partially purified calf uterine estrogen receptor (ER) to a 38-base pair estrogen responsive element (ERE) consensus sequence, containing the inverted repeat 5'-GGTCAnnnTGACC-3', was measured in vitro. The ERE sites were inserted as single or multiple tandem copies in a plasmid vector [p GEM-7Zf(+)]. Results showed that one dimeric ER can interact with one ERE, and steric constraints do not inhibit binding of ER to adjacent EREs. Molybdate-stabilized monomeric (4S) ER did not bind to EREs. ER bound to single and tandem double EREs with Kd values of 0.24 and 0.23 nM, respectively. When the plasmid contained three or more tandem copies of the ERE, ER bound in a cooperative manner, as indicated by convex Scatchard plots and Hill coefficients greater than 1.5. To determine those characteristics of the consensus sequence that are important for maximal high-affinity ER binding, ten variant ERE oligomer sequences were synthesized and cloned into pGEM-7Zf(+) as single copies or as four copies in tandem. ER binding affinity was maximal for the consensus ERE and was reduced for variants containing one or two nucleotide changes in the inverted repeat. The number of nucleotides separating the inverted repeat in the ERE was critical for high-affinity ER binding. Certain sequence-variant EREs when cloned as single copies bound less ER compared to the consensus ERE, yet when cloned as four tandem copies, ER binding displayed cooperativity by Scatchard and Hill analyses. Results demonstrate that cooperative interactions noted in vivo by others are present when measured in vitro. Results strongly imply that the number, spacing, and nucleotide sequence of EREs could precisely control the amount of ER binding to estrogen-responsive genes.

Base Sequence↗

PubMed1990

A microtiter well assay for quantitative measurement of estrogen receptor binding to estrogen-responsive elements.

Reproducible, rapid measurement of estrogen receptor (ER) binding to DNA was accomplished in microtiter wells treated so that ER-DNA complexes or DNA bound in preference to free ER. Mixtures of 35S-labeled DNA and [3H]estrogen-charged ER ([3H]ER), incubated to equilibrium in microfuge tubes, were transferred to microtiter wells previously treated with histone followed by gelatin. After binding of the DNA or ER-DNA complex to the treated wells, free ER was removed by washing. Radioactivity retained in each well was measured by placing individual wells from snap-apart microtiter plates directly in scintillation fluid. Binding of DNA was saturable, and ER-DNA complex binding was complete within 2 h at 4 C. The use of 35S-labeled DNA and [3H]ER allowed stoichiometric determination of ER bound to DNA. The amount of ER specifically bound to a consensus estrogen-responsive element (ERE) containing the inverted repeat GGTCAgagTGACC was determined by comparing ER bound to plasmid containing or lacking the ERE. At saturating concentrations of ER, plasmids bearing one, two, and four EREs in tandem bound approximately one, two, and four dimeric ER molecules, respectively. Scatchard analysis of saturation binding data revealed a Kd of 0.15 nM for specific ER binding to a single ERE site. Thus, the assay detects ER retaining both DNA-binding and estrogen-binding functions. ER complexed with DNA in the well was also detected using a monoclonal antibody specific for the receptor. Simple modifications of this method would allow study of other DNA-protein interactions.

Animals↗

PubMed1989

Rapid purification of the estrogen receptor by sequence-specific DNA affinity chromatography.

Rapid purification of calf uterine estrogen receptor (ER) to near homogeneity has been accomplished by use of sequence-specific DNA affinity resin. Very high selectivity for the estrogen receptor is achieved through the use of DNA-Sepharose containing eight tandem copies of a consensus estrogen response element (ERE) DNA sequence. The highly purified ER prepared by this new scheme may be labeled economically with ligands of high specific activity. This purification scheme selects for intact receptors retaining function in both estrogen-binding and DNA-binding domains. Purified receptor has an electrophoretic mobility consistent with a molecular weight of 68,000, sediments as a 5S species on sucrose gradients, and reacts with antibody specific to the human estrogen receptor.

Animals↗

PubMed1988

Properties of a high-affinity DNA binding site for estrogen receptor.

It has been shown previously that a short sequence from the 5' regulatory region of the Xenopus laevis vitellogenin gene A2, when appropriately placed, can confer estrogen responsiveness to another gene. Using the Xenopus sequence and similar sequences from the 5' regulatory regions of other estrogen-responsive genes, we derived a consensus sequence 38 nucleotides long. The sequence contains an inverted repeat (5' C-A-G-G-T-C-A-G-A-G-T-G-A-C-C-T-G 3') and an A/T-rich region. Plasmids carrying a single copy of the sequence bound 3-fold-more partially purified estrogen receptor (ER) than did control plasmids when assayed by gel filtration. Maximum specificity for ER binding occurs at 100-150 mM ionic strength and pH 7.5-8.0. Plasmids carrying multiple copies of the sequence bound correspondingly more ER. The dissociation constant for ER bound to the sequence is 0.5 nM. This value is lower by a factor of about 400 than the dissociation constant for ER bound to an equivalent length of plasmid DNA. Portions of the consensus sequence were evaluated for binding efficiency. Plasmids containing the inverted repeat alone bound ER, though less efficiently than did plasmids containing the entire sequence. The A/T-rich region alone was ineffective in binding ER. Linearization of the plasmid DNA did not enhance specific binding efficiency for ER. This model system represents an effective tool for characterization of ER binding to DNA sequences involved in the regulation of gene expression.

Animals↗

PubMed1985

An analysis of the decrease in the assayed level of charged bovine estrogen receptor observed at physiological ionic strength.

Lower assayed levels of heifer uterine estrogen receptor (ER) occur at physiologic ionic strength when ER is separated from [3H]estradiol by Dextran-coated charcoal treatments, or by gel filtration on Sephadex or polyacrylamide resins. The assayed level of charged ER in buffers containing 150-200 mM ionic strength is approximately one-half that of ER levels assayed in buffers either at 0-50 or 400-450 mM ionic strength. Treatment of ER with trypsin or molybdate eliminates this observed reduction. Evidence is presented that the decrease results from a preferential adsorption of ER to the assay resins at 150-200 mM ionic strength. This adsorption is likely to be mediated by a hydrophobic region of the ER, which is removed by trypsin cleavage.

Acrylic Resins↗