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D P Edwards

Publications and source records attributed to D P Edwards.

At least 109 records · Page 6Linked to original sources
PubMed1989

Human progesterone receptor complexed with the antagonist RU 486 binds to hormone response elements in a structurally altered form.

Structural and functional properties of human progesterone receptors (PR) bound with the antiprogestin, RU 486, and the progestin agonist, R5020, were compared in order to identify receptor mechanisms responsible for the inability of RU 486 to activate the transcriptional capacity of receptors. RU 486 interaction with human PR did not inhibit receptor transformation as assessed by dissociation of nontransformed 8-10S oligomeric receptors (in vitro and in vivo) and by tight binding of PR to nuclei/chromatin in whole cells. Assays based on immunoprecipitation of PR-DNA complexes with an antibody to human PR and gel retardation were used to analyze the effect of RU 486 on receptor binding to the hormone response element (HRE) of the mouse mammary tumor virus (MMTV). RU 486 did not impair PR recognition of the MMTV HRE. Quantitative affinity constants and kinetic parameters of PR binding to these specific DNA sites were similar for receptors complexed with either agonist or antagonist. However, PR-RU 486 complexes exhibited an altered sedimentation rate on sucrose gradients and a faster mobility when bound to the MMTV HRE as assessed by gel retardation. These results indicate that human PR transformed by RU 486 exhibit no impairment in binding to specific DNA sites of target genes, but when bound to DNA assumes a structural form different from that of the receptor-agonist complexes to activate transcription results from this structural alteration in PR, which does not permit protein-protein interactions required for receptor-mediated induction of gene transcription.

Blotting, Northern↗
PubMed1989

Human progesterone receptor binding to mouse mammary tumor virus deoxyribonucleic acid: dependence on hormone and nonreceptor nuclear factor(s).

Using crude progesterone receptor preparations from T47D human breast cancer cells, we show by immunoprecipitation assay that receptor specifically and with high affinity recognizes the hormone response element (HRE) of the mouse mammary tumor virus (MMTV). The use of crude preparations minimizes alterations of receptors or loss of associated factors that may occur during purification. Specific binding was obtained at 1:1 molar ratios of receptor to DNA, and HRE sequences are recognized with an affinity at least 3 orders of magnitude greater than nonspecific DNA. We have compared the DNA-binding activities of different forms of progesterone receptors. The unliganded 8S cytosol receptor had low but detectable binding activity for MMTV DNA. Addition of hormone to cytosol produced a small but consistent 2.5-fold increase. In vitro methods of transforming cytosol receptors from an 8S to a 4S species failed to increase DNA-binding further. By contrast, 4S receptors bound by R5020 in whole cells and extracted from nuclei by salt, displayed a substantially higher (average, 11-fold) binding activity than an equal number of unliganded cytosol receptors. The dissociation constants for cytosol and nuclear receptor binding to MMTV DNA were similar (approximately 2 x 10(-9) M). Thus, nuclear receptors possess a higher capacity for binding to specific recognition sequences. These results suggest that hormone or a hormone-dependent mechanism increases the intrinsic DNA-binding activity of receptors independent of receptor transformation from 8S to 4S. Further experiments indicate that a nonreceptor activity in nuclear extracts can increase the sequence-specific DNA-binding activity of cytosol receptors. This activity is present in both T47D cells and receptor-negative MDA-231 cells. We conclude that the higher DNA-binding activity of the nuclear receptor-hormone complex is due in part to receptor interaction with other nuclear proteins or factors. Such interactions may function to maintain receptors in a disaggregated active complex or to stabilize their binding to specific DNA sites.

Animals↗
PubMed1989

A quantitative comparison of dual control of a hormone response element by progestins and glucocorticoids in the same cell line.

Progesterone receptor-containing T47D human breast cancer cells are responsive to progestins but fail to respond to other steroid hormones, in particular dexamethasone, because they have no measurable levels of receptors for estrogens, androgens, or glucocorticoids. To quantitatively study dual responsiveness of the mouse mammary tumor virus (MMTV) promoter to progestins and glucocorticoids, we have stably transfected T47D cells with a glucocorticoid receptor (GR) expression vector. A cloned derivative (A1-2) was isolated that expresses a normal, full length GR, as assessed by steroid binding and Western immunoblot with a monoclonal anti-GR antibody. Moreover, GR is expressed at levels (80,000-100,000 molecules per cell) comparable to the high levels of endogenous progesterone receptor (200,000 molecules per cell). In A1-2 cells transiently transfected with an MMTV-chloramphenicol acetyl transferase reporter gene, induction by glucocorticoid was substantially greater (5-fold) than induction mediated by progestins. These results suggest that glucocorticoids may be the primary regulator of MMTV.

Breast Neoplasms↗
PubMed1988

Immunocytochemical localization of progesterone receptors in breast cancer with anti-human receptor monoclonal antibodies.

Monoclonal antibodies (MAbs), recently produced against human progesterone receptors (PR), were used for immunocytochemical localization of PR. The specificity of the immunocytochemical assay for PR was demonstrated by incubation with control MAbs, preabsorption of MAbs with highly purified human PR, and by the cell and tissue distribution of the immunostaining reaction. With human breast cancer cell lines, immunoreactivity was confined to cells that contain PR by steroid-binding assay. Moreover, immunostaining was induced by estradiol in estrogen-responsive cells, MCF-7 and ZR-75-1. In a preliminary study with 33 breast carcinomas, a good correspondence was obtained between immunocytochemical staining and PR content assessed by conventional steroid-binding assay. Immunoperoxidase localization was also obtained with other human target tissues. In normal breast and benign breast disease, immunoreactivity was observed with nuclei of ductal epithelial cells and hyperplastic epithelium. In uterus, immunostaining of endometrium was localized to nuclei of stromal and glandular epithelial cells and in myometrium to nuclei of smooth muscle cells. The effect of the progestin agonist, R5020, and antagonist, RU 486, on PR localization was investigated with the PR-rich T47D human breast cancer cell line. In the absence of hormone, immunostaining was exclusively nuclear. This was true under a number of cell culture conditions designed to eliminate endogenous progestins from the culture medium. Exclusive nuclear localization of PR was not due to a failure of the MAbs to recognize unoccupied PR, since each MAb bound equally well in vitro with different receptor forms. These included liganded and unliganded cytosol PR, molybdate stabilized PR, and nuclear-transformed receptors. Nor was failure to detect cytoplasmic staining due to a selective destruction or loss of unoccupied PR from the cytoplasmic compartment as a result of cell fixation. This was assessed by dot blot immunoassay of PR antigen distribution in subcellular fractions of fixed and unfixed cells. Continuous exposure of cells to R5020 resulted in a transient (30-60 min) increase in nuclear staining intensity (without change in cytoplasmic reactivity), followed by a progressive decline in immunoreactivity. By 24 h of R5020 treatment, the vast majority of cells displayed no immunostaining reaction. These immunocytochemical data are consistent with progestins down regulating their own receptors due to a loss in cellular PR content and not to inactivation of receptors.(ABSTRACT TRUNCATED AT 400 WORDS)

Antibodies, Monoclonal↗
PubMed1988

Steroid hormone-dependent interaction of human progesterone receptor with its target enhancer element.

We investigated the requirement of steroid hormone for the specific binding of progesterone receptor to its cognate progesterone responsive element (PRE) in cell-free experiments. We prepared unfractionated nuclear extracts from human breast cancer (T47D) cells which are rich in progesterone receptors and used a gel retardation assay to monitor receptor-DNA complex formation. Exposure of receptor to either progesterone, R5020, or the antiprogestin RU38 486 in vivo or in vitro led to the formation of two protein-DNA complexes (1 and 2) which were not detected in nuclear extracts unexposed to hormone. Similar treatment with cortisol or estradiol failed to induce the formation of these complexes. The complexes were specific for PRE, since they could be competed efficiently in binding competition experiments by oligonucleotides containing PRE. A monoclonal antibody which recognizes both A and B forms of human progesterone receptor, interacted with both complexes 1 and 2 and shifted them to slower migrating forms. Another antibody which only recognizes the B form interacted with only complex 1 but not with complex 2, establishing that the complexes 1 and 2 were indeed formed by progesterone receptor forms B and A, respectively. We conclude from the above studies that in vivo or in vitro treatment of nuclear progesterone receptor with either progesterone or R5020 or RU38 486 alone can lead to detection of high affinity complexes formed between the PRE and the receptor present in unpurified nuclear extracts.

Breast Neoplasms↗
PubMed1987

Immunologic analysis of human breast cancer progesterone receptors. 1. Immunoaffinity purification of transformed receptors and production of monoclonal antibodies.

A monoclonal antibody (MAb), designated PR-6, produced against chick oviduct progesterone receptors [Sullivan, W. P., Beito, T. G., Proper, J., Krco, C. J., & Toft, D. O. (1986) Endocrinology (Baltimore) 119, 1549-1557] cross-reacts with the Mr 120,000 human B receptors. An immunomatrix prepared with PR-6 was used to purify progesterone receptors (PR) from T47D human breast cancer cells. Single-step immunoaffinity chromatography results in enrichment of B receptors (identified by immunoblot with PR-6 and by photoaffinity labeling with [3H]promegestone) to a specific activity of 1915 pmol/mg of protein (or 23% purity) and with 27% yield. Purity and yields as judged by gel electrophoresis and densitometric scanning of the B protein were approximately 1.7-fold higher due to partial loss in hormone binding activity at the elution step. A second purification step by diethylaminoethyl chromatography gives further enrichment to 3720 pmol/mg of protein (or 44% purity) to yield essentially two proteins, 120-kilodalton (kDa) B receptors and a 76-kDa non-steroid binding protein, each in approximately equivalent amounts. B receptors purified under these conditions are transformed and biologically active. They were maintained as undegraded 120-kDa doublets and retained both hormone and DNA binding activities. Isolated B receptors were free of the 90-kDa non-steroid binding protein observed to be associated with 8S untransformed receptors in other systems and were free also of the non-hormone binding 105-108-kDa B antigen described previously to copurify with chick PR. These purified B receptors were used as immunogen for production of four monoclonal antibodies against human PR. Three of the MAbs, designated as B-30 (IgG1), B-64 (IgG1), and B-11 (IgM), are specific for B receptors. The fourth MAb, A/B-52 (IgG1), reacts with both A and B receptors. The IgG MAbs are monospecific for human PR since they recognize and absorb native receptor-hormone complexes, displace the sedimentation of 4S receptors on salt containing sucrose gradients, and, by immunoblot assay of crude T47D cytosol, react only with receptor polypeptides. Although mice were injected with B receptors only, production of A/B-52 which recognized both A and B receptors provides evidence that these two proteins share regions of structural homology. These new MAbs are valuable reagents for further studies of human receptor structure and function and for clinical immunodetection of PR in breast tumors.

Animals↗
PubMed1987

Immunologic analysis of human breast cancer progesterone receptors. 2. Structure, phosphorylation, and processing.

We have used a monoclonal antibody (MAb) directed against chick oviduct progesterone receptors (PR), that cross-reacts with human PR, to analyze PR structure and phosphorylation. This MAb, designated PR-6, interacts only with B receptors (Mr 120,000) of T47D human breast cancer cells; it has no affinity for A receptors (Mr 94,000) or for proteolytic fragments from either protein. The antibody immunoprecipitates native B receptors and was used to study the structure of native untransformed 8S and transformed 4S receptors, using sucrose density gradient analysis, photoaffinity labeling, and gel electrophoresis. On molybdate-containing low-salt gradients, PR-6 complexes with 8S B receptors, causing their shift to the bottom of the gradient while A receptors remain at 8 S. Therefore, A and B receptors form separate 8S complexes, and we conclude that A and B do not dimerize in the holoreceptor. Similar gradient studies using salt-containing, molybdate-free buffers show that there are two forms of salt-transformed 4S receptors, comprising either A proteins or B proteins, suggesting that A and B are also not linked to one another in transformed PR. The independence of A- and B-receptor complexes was confirmed by the finding that purified, transformed B receptors bind well to DNA-cellulose. Since PR-6 cross-reacts with nuclear PR, it was used to analyze nuclear PR processing--a down-regulation step associated with receptor loss as measured by hormone binding. Insoluble nuclear receptors and soluble cytosol receptors were measured by immunoblotting following treatment of T47D cells for 5 min to 48 h with either R5020 or progesterone. From 8 to 48 h after R5020 treatment, immunoassayable receptors decreased in nuclei and were not recovered in cytosols. Nuclear receptors also decreased after progesterone treatment but replenished in cytosols between 8 and 24 h after the start of treatment. Thus, processing involves a true loss of nuclear receptor protein, and not just loss of hormone binding activity, and occurs after progesterone or R5020 treatment. This loss is chronic, however, only in R5020-treated cells. Additional studies focused on the covalent modifications of receptors. We previously described shifts in apparent molecular weight of nuclear PR following R5020 treatment using in situ photoaffinity labeling. To show whether these shifts can be explained by receptor phosphorylation, untreated cells and hormone-treated cells were metabolically labeled with [32P]orthophosphate, and the B receptors were isolated by immunoprecipitation with PR-6 and analyzed by sodium dodecyl sulfate (SDS) gel electrophoresis.(ABSTRACT TRUNCATED AT 400 WORDS)

Breast Neoplasms↗
PubMed1987

An immunotoxin cytotoxic for breast cancer cells in vitro.

A potent immunotoxin was formed by conjugating the murine monoclonal antibody 323/A3 to the A chain of ricin. The 323/A3 antibody recognizes an antigen expressed by most human breast cancers. When binding of 323/A3 is examined by enzyme-linked immunosorption assay, three human breast cell lines displayed strong binding, whereas two human breast cell lines and three non-breast cell lines displayed little or no binding. When the cell lines were tested at a concentration of 0.1 microgram/ml, those cell lines which displayed an abundance of antigen by enzyme-linked immunosorption assay were most sensitive to the effects of the 323/A3 immunotoxin. On the other hand, cell lines which displayed little or no antigen by enzyme-linked immunosorption assay were not inhibited by the immunotoxin at this concentration. Further examination of the effects of immunotoxin concentration on protein synthesis confirmed the sensitivity of those cell lines rich in the 323/A3 antigen over a broad dose range. Similarly, three cell lines which displayed little of the 323/A3 antigen demonstrated little inhibition of protein synthesis with various concentrations of 323/A3 immunotoxin. However, two cell lines which displayed little antigen were intermediate in their sensitivity to the 323/A3 immunotoxin. Preclinical evaluation of immunotoxins as potential therapeutic agents will require accurate and sensitive screening of a wide variety of cell types. The 323/A3 remains of interest in studying the effects of immunotoxin in a defined in vivo model system.

Animals↗
PubMed1986

Monoclonal antibody identification and characterization of a Mr 43,000 membrane glycoprotein associated with human breast cancer.

A monoclonal antibody (323/A3) with a high degree of selectivity for binding to breast cancer cells was produced by immunization of mice with MCF-7 human breast cancer cells. The antigen recognized by 323/A3 on MCF-7 appears to be surface localized, and by enzyme-linked immunosorbent assay, the antibody was found to bind strongly with four of six breast cancer cell lines examined while no binding was detectable with nonbreast cancer cell lines. In vivo distribution of the 323/A3 antigen was screened by immunoperoxidase staining of formalin-fixed paraffin sections of normal human tissues and tumors. Among breast tissues, positive staining was detected with 75% (6 of 8) of metastatic lymph nodes, 59% (76 of 128) of primary breast tumors, 20% (13 of 63) of benign breast lesions, and 0% (0 of 10) of normal breast. No immunostaining was detected with a large variety and number of other normal human tissues with the exception of staining observed with epithelium of normal colon. Antigen distribution appears not to be disease specific, since positive staining was also observed with adenocarcinomas other than breast. The antigen recognized by the 323/A3 antibody was identified by Western blot analysis as a Mr 43,000 protein. The glycoprotein nature of the antigen was demonstrated by its binding to concanavalin A, specific elution with sugar, and immunoprecipitation of a Mr 43,000 radiolabeled protein from extracts of MCF-7 cells after pulse labeling with [3H]glucosamine. The 323/A3 antigen appears to be the same Mr 43,000 protein in cell lines as in breast tumors in vivo. Based on a comparison with the molecular weights of other known tumor-associated antigens and with their immunocytochemical tissue distribution, the Mr 43,000 glycoprotein described here represents a tumor-associated antigen previously undescribed in breast cancer or in other tumors. Since the Mr 43,000 glycoprotein is present on the surface of most breast cancer cells and is either absent or expressed at very low levels in most normal tissues including normal breast, the monoclonal antibody described here may have potential applications in diagnosis and management of breast cancer.

Antibodies, Monoclonal↗
PubMed1985

Immunologic detection of a protein homologous to chicken progesterone receptor B subunit.

A monoclonal antibody described previously by us (Edwards, D. P., Weigel, N. L., Schrader, W. T., O'Malley, B. W., and McGuire, W. L. (1984) Biochemistry 23, 4427-4435) was used to study progesterone receptor B subunits of chick and hen oviduct. We find that the antibody does not recognize the form of receptor B able to bind [3H]progesterone in vitro. Rather, it reacts exclusively with a homologous protein of the same molecular weight, termed B antigen. The antigen is present in both immature estrogen-treated chicks and in egg-laying hens. This antigen is indistinguishable from the hormone-binding receptor species (termed receptor B) as shown by peptide mapping techniques using either Staphylococcus aureus V8 protease or trypsin. The B antigen and the hormone binder can be resolved by ion-exchange chromatography. Sedimentation velocity data show that the two proteins are present in distinct, separable cytosolic entities. The functional relationship between the two proteins has not been established.

Age Factors↗
PubMed1985

The presence of an estrogen-regulated protein detected by monoclonal antibody in abnormal human endometrium.

The cellular localization of a 24,000 (24K) mol wt protein was evaluated by monoclonal antibody immunocytochemistry in atrophic and persistent proliferative endometrium as well as in various forms of hyperplastic and neoplastic human endometria. 24K was chiefly located in the cytoplasm of ciliated cells and their precursor forms, clear cells. It was absent in atrophic, resting, and regressive forms of cystic glandular hyperplasia. Persistent proliferative endometria with a low degree of proliferation has occasional 24K immunostained cells, whereas those with active proliferation contained intense 24K immunostaining. A similar pattern characterized the active forms of cystic glandular hyperplasia. The immunostaining reaction decreased in adenomatous and atypical adenomatous hyperplasia as well as in well differentiated adenocarcinomas. In poorly differentiated carcinomas, 24K protein was virtually absent. These findings suggest that 24K protein is estrogen and ciliated cell related, and endometrial ciliogenesis and 24K protein are morphological and biochemical markers, respectively, of the estrogenic endometrial response. Their increase and decrease in hyperplastic and neoplastic endometria support the concept that early hyperplasia is highly sensitive to estrogenic stimulation, whereas with increasing architectural and cytologic atypia, the estrogenic response decreases as a reflection of growing cell populations that are independent of estrogenic influence. Immunostaining with 24K is not sensitive enough to discriminate estrogen-independent cells of atypical adenomatous hyperplasia from those of early, well differentiated adenocarcinoma.

Adult↗
PubMed1985

Immunological detection of Chinese hamster ovary cells expressing a multidrug resistance phenotype.

A monoclonal antibody (IgG1) has been prepared that specifically detects Chinese hamster ovary cells expressing a multidrug-resistant phenotype. This antibody recognizes the membrane P-glycoprotein (Mr 170,000) associated with drug resistance as determined by enzyme-linked immunosorbent assay with purified P-glycoprotein and by Western blot analysis of cell extracts from drug-resistant and drug-sensitive cells. By immunofluorescence methods, the antibody also reacts strongly with viable and ether:ethanol-fixed resistant cells but does not react with the parent drug-sensitive cell line. Thus, this antibody can bind with live cells allowing discrimination by immunohistochemistry between drug-resistant and drug-sensitive Chinese hamster ovary cells.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1984

Structural analysis of chicken oviduct progesterone receptor using monoclonal antibodies to the subunit B protein.

Two monoclonal antibodies against the B subunit (Mr 108 000) of chick oviduct progesterone receptor (PgR) were produced by immunizing rats and fusing spleen cells with NS-1 mouse myeloma cells. The hybridoma lines designated 9G10 and 3E8 produce rat IgG2a and IgG2b, respectively. Antibody-receptor interactions were demonstrated under protein denaturing conditions. Previous studies by Weigel et al. [Weigel, N. L., Tash, J. S., Means, A. R., Schrader, W. T., & O'Malley, B. W. (1981) Biochem. Biophys. Res. Commun. 102, 513-519] have shown that chick PgR can be phosphorylated in vitro. Both antibodies, 9G10 and 3E8, were shown to displace partially denatured 32P-labeled PgR from its characteristic 4S position on high salt sucrose density gradients to a form with a higher sedimentation coefficient. Further specificity and sensitivity were demonstrated by protein immunoblotting experiments. In partially purified as well as electrophoretically pure receptor B subunit preparations antibodies reacted with the Mr 108 000 receptor B band. By immunoblot assay 9G10 was 20-fold more sensitive than 3E8, the former detecting down to 5 ng of receptor and the latter 100 ng. Because of its sensitivity 9G10 was able to detect the Mr 108 000 receptor as a single band in a crude oviduct fraction and did not cross-react with any other contaminating proteins. Receptor antigenic determinants were localized by immunoblot assay of receptor proteolytic digests.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1984

Varicella-zoster virus-specific gp140: a highly immunogenic and disulfide-linked structural glycoprotein.

A 140,000-dalton disulfide-linked glycoprotein (gp140) specified by varicella-zoster virus (VZV) in infected cultured cells was identified and precipitated by two murine monoclonal antibodies (VZ-151 and VZ-158). When analyzed under reducing conditions by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, gp140 was cleaved predominantly into a 66,000 lower-molecular-weight product (gp66). This protein was classified as a viral structural component, since it was observed in the polypeptide profile of metrizamide gradient-purified enveloped virions. By immunofluorescence analyses with a monoclonal antibody probe, gp140 expression was documented to be highly conserved both within cultured cells inoculated with homologous (VZV-Oka) and heterologous (VZV-32) strains and in infected human tissues from chicken pox and zoster patients. That the glycoprotein was highly immunogenic was confirmed by the presence of high-titer anti-gp140 antibody in the sera of both hyperimmunized laboratory animals and naturally infected humans. Temporally, the humoral response to gp140 following primary VZV infection preceded that against the other viral glycoproteins. These studies describe, therefore, an immunogenic, disulfide-linked viral structural glycoprotein, which must be included among the other five previously described VZV-specific fucosylated species--gp118, gp98, gp62, and gp45.

Antibody Formation↗
PubMed1984

Estrogen-induced 24K protein in MCF-7 breast cancer cells is localized in granules.

We have previously reported the production of monoclonal antibodies which detect, by immunohistochemistry, an estrogen-induced protein of molecular weight 24,000 daltons (24K). This protein, of unknown function, has been detected in: a) estrogen receptor-positive breast cancer cell lines but not in receptor-negative lines; b) several human normal estrogen target organs; and c) certain human carcinomas, including breast tumors. To examine the subcellular localization of this 24K estrogen-induced protein, we have done immunohistochemical studies at light and electron microscopic levels using a human breast tumor cell line (MCF-7) grown in vitro and also in nude mice in vivo. MCF-7 cells grown in the ascites fluid of nude mice and processed for paraffin sections showed a defined polarity, and the 24K protein was localized in the apical cytoplasm of the cells. After cytocentrifugation, MCF-7 cells grown in vitro displayed 24K protein mainly confined to large cytoplasmic granules. The presence of 24K protein in cytoplasmic granules was also seen by immunoelectronmicroscopy in MCF-7 cells grown both in vitro and in vivo. The granules had different sizes, shapes, and 24K immunostaining intensity. The morphological evidence suggests that the 24K estrogen-induced protein is secreted from the cells.

Animals↗
PubMed1984

Estrogen-regulated protein in human breast cancer.

An estrogen-regulated cytoplasmic protein has been purified from MCF-7 human breast cancer cells with anion exchange and monoclonal antibody affinity chromatographies. The purified protein has a monomeric mol. wt of 28,000 and isoelectric species with pI's between 5.9 and 6.0. Amino acid analysis indicates the protein is acidic, is probably hydrophilic, and contains unusually low amounts of methionine and half cystine. The monoclonal antibodies are of the IgG1 subclass, exhibit a high affinity for the 28K protein, and recognize an antigenic site that is stable to sodium dodecyl sulfate (SDS) gel electrophoresis and electrophoretic transfer techniques. These monoclonal antibodies have been used to detect 28K in certain human breast tumors.

Amino Acids↗
PubMed1984

Molecular cloning of a cDNA for the chicken progesterone receptor B antigen.

A cDNA for the chicken progesterone receptor B subunit antigen (Mr, 108,000) has been isolated from a cDNA library prepared from size-selected chicken oviduct poly(A)+RNA. A specific monoclonal antibody raised against hen progesterone receptor B subunit (alpha PR-B) was used to screen the library. Recombinant clones reacting with the antibody by virtue of antigen expression were used in hybrid-selected translation. A single clone, pPRB-1, hybridized specifically to a mRNA that yielded a Mr 108,000 protein when translated in vitro and which was immunoprecipitable by the alpha PR-B antibody. This cDNA represents a 470-base-pair portion of the PR-B nucleotide sequence. Additional clones have been subsequently isolated from the recombinant library using the insert from pPRB-1 as a specific probe. A mRNA size of approximately 3000 nucleotides was determined for the chicken progesterone receptor B subunit by formaldehyde/agarose gel electrophoresis and blot hybridization using pPRB-1 as a probe. Preliminary studies show that withdrawal of hormone from chickens treated chronically with estrogen leads to a dramatic decrease in the cellular RNA concentration of receptor B, indicating that target tissue levels of receptor B RNA are under hormonal control.

Animals↗