Biomedical subjects

D F Gordon

Publications and source records attributed to D F Gordon.

At least 19 recordsLinked to original sources

PubMed1992

Structure of the rat gonadotropin releasing hormone (rGnRH) gene promoter and functional analysis in hypothalamic cells.

The gonadotropin releasing hormone (GnRH) gene encodes a protein which plays a critical role in mammalian reproductive physiology. Its expression is predominantly restricted to the hypothalamus although it has also been described in the placenta. To begin to determine the promoter elements important for tissue specific expression and to examine the mechanisms of developmental and hormonal regulation of the rat GnRH (rGnRH) gene, we cloned the rGnRH gene from a rat liver genomic DNA library. The nucleotide sequence of greater than 3 kb of 5'-flanking region was determined. The transcriptional initiation site in rat hypothalamic tissue and a mouse hypothalamic cell line were mapped by primer extension analysis and found to be different. In addition, transient transfection studies demonstrated that multiple regions of the distal promoter are important for tissue specific and basal promoter activity in hypothalamic cells. Furthermore, in these cells a potent activation region resides between -3026 and -1031 bp and suppressor region between -1031 and -903 bp upstream of the transcriptional start site. We conclude that different portions of the 5'-flanking region, which are activating and suppressing in nature, are critical for hypothalamic expression of the rGnRH gene.

Animals↗

PubMed1992

Estrogen negatively regulates rat gonadotropin releasing hormone (rGnRH) promoter activity in transfected placental cells.

To dissect the functional architecture of the rat gonadotropin releasing hormone (GnRH) gene promoter and its regulation by estrogen, gene transfer studies were performed in a placental cell line. 5'-Deletional constructs demonstrated that cis-acting elements important for rGnRH promoter activity in placenta were contained within a region of the proximal promoter between -73 and -16 bp upstream of the transcription initiation site. In addition, an inhibitory region was found from -903 to -424. Deletion of sequences to -424 which removed the inhibitory region, produced a promoter fragment which exhibited a 50% inhibition of GnRH promoter activity in the presence of estrogen (E) and cotransfected estrogen receptor. Negative regulation by E was retained in constructs deleted to -73 bp. We conclude that sequences important for placental cell expression and estrogen regulation of rGnRH lie within the region from -73 to -16. In addition, potential repressor sequences active in placental cells are present between -903 to 424.

Animals↗

PubMed1992

A cell line that produces the glycoprotein hormone alpha-subunit contains specific nuclear factors similar to those present in thyrotropes.

A unique characteristics of thyrotrope-specific gene expression is the coordinated expression and regulation of the alpha- and beta-subunits of TSH. A cell line (alpha TSH) derived from the transplantable mouse thyrotropic tumor MGH101A, which no longer expresses the TSH beta-subunit gene but continues to secrete large amounts of alpha-subunit, was used as a model to study alpha-subunit gene expression independent from the TSH beta-subunit gene and was compared with the expression in TSH-secreting TtT97 tumors. Transient transfection studies showed a striking similarity in the activity of 5' deletions of the mouse alpha-subunit gene promoter in both alpha TSH and TtT97 cells and localized two regions important for expression that spanned 100 base pairs, from -480 to -417 and from -417 to -381. These regions were found to have no activity in nonthyrotrope pituitary GH4 cells and L-cell fibroblasts. Analysis of the alpha-subunit 5' flanking DNA interactions with alpha TSH and TtT97 nuclear extracts showed two DNase I protected sequences, from -474 to -452 and from -447 to -400, both of which colocalized with the functionally important regions. Gel retardation analysis demonstrated the specificity of these interactions, and a similar migration of the DNA-protein complexes suggested that protein factors were similar in the two cell types. We conclude that the nuclear factors necessary for alpha-subunit expression in thyrotropes are retained in alpha TSH cells. Moreover, since alpha TSH cells do not express the TSH beta-subunit gene, the factors that determine the expression of the alpha-subunit may not be sufficient for TSH beta-subunit gene expression.

Animals↗

PubMed1991

Limited impact of testicular self-examination promotion.

Analysis of data obtained through a survey of 415 men in New Orleans, Louisiana, and Rochester, New York, indicates that the transfer of testicular self-examination (TSE) skills from the medical community to the public has been relatively ineffective. Only two of every 100 respondents reported monthly self-examination performed at the correct time and with the proper method. The findings suggest that, until the transfer of self-detection skills is improved, one cannot assess the efficacy of the technology itself. Problems impeding such improvement are reviewed.

Adolescent↗

PubMed1991

Isolation and characterization of mouse complementary DNAs encoding alpha and beta thyroid hormone receptors from thyrotrope cells: the mouse pituitary-specific beta 2 isoform differs at the amino terminus from the corresponding species from rat pituitary tumor cells.

Thyroid hormones (T3) and their receptors (TR) play a critical role in the function of the pituitary gland, particularly in thyrotropes, where they regulate expression of the alpha- and beta-subunits of TSH. Since the pituitary gland is composed of several cell types, we undertook a characterization of TR subtypes in a murine thyrotropic tumor (TtT-97), an excellent model in which to study thyroid hormone action in thyrotropes. We screened a thyrotrope cDNA library with rat TR alpha 1 and TR beta 1 cDNA probes and isolated cDNAs encoding the mouse TR alpha 1 and TR beta 1 isoforms as well as a partial clone corresponding to the non-T3 binding carboxy-terminal alpha 2 variant. The polymerase chain reaction was used to amplify additional cDNAs for the specific 5' domains of the mouse TR beta 1 and the pituitary-specific TR beta 2 amino-terminal variant. Using hybridization probes that discriminate between the alpha and beta isoforms and their variants, we demonstrated that thyrotropes contain TR alpha 1 and alpha 2 mRNAs as well as transcripts encoding Rev-erbA, which arise by transcription from the opposite strand of the TR alpha gene. In thyrotropes, the ratio of alpha 2 to TR alpha 1 mRNA levels more closely resembled the distribution in mouse brain than that in heart, where the mRNA levels of TR alpha 1 and alpha 2 are comparable. TR beta 1 and TR beta 2 mRNAs were detected in thyrotropes and were of similar size (approximately 6.4 kilobases). Despite the almost complete conservation between the rat and mouse TR beta 1 sequences at the protein level, the mouse and rat TR beta 2-specific N-terminal domains were less conserved, and the mouse protein was shorter by 39 amino acids at the N-terminus. Of the receptor species, only the mRNA encoding the TR beta 2 isoform, which was restricted to thyrotropes, was decreased by T3 treatment, although the mRNA for the alpha 2 variant was also reduced by T3 in thyrotropes and heart tissue. Levels of TR beta 1 mRNA were not changed in liver, but were increased in thyrotropic tumors and also somewhat in brain, an organ that is not responsive to T3 by classical criteria.

Amino Acid Sequence↗

PubMed1990

T47DCO cells, genetically unstable and containing estrogen receptor mutations, are a model for the progression of breast cancers to hormone resistance.

We postulate that one mechanism for the progression of breast cancers to hormone resistance involves the acquisition of mutant estrogen receptors (ER)4 by genetically unstable cell subpopulations. The T47D human breast cancer cell line may be a model for such progression, having sublines that are ER positive and estrogen responsive, ER positive and estrogen resistant, or ER negative. Also, T47D cells can be either hyperdiploid (HD) or hypertetraploid (HT) or persistently alternate between these states. T47DCO cells are a HD and ER-positive, but estrogen-resistant, subline of T47D cells that undergoes spontaneous tetraploidization. Such a stable variant, designated T47Dv, is 85% HT (Cancer Res., 49: 3943, 1989). We now show that single-cell clones derived from the mixed HD/HT T47Dv can be either HD or HT, and can be either estrogen responsive or estrogen resistant, for growth and for progesterone receptor regulation. To begin the study of ER in this model system of T47DCO and their derivatives, we have generated complementary DNA libraries from the parental HD T47DCO cells and have isolated three ER complementary DNA mutants. These include two frame-shift/termination mutants that would encode ERs truncated in the DNA-binding domain and in the hormone-binding domain and a third mutant with a large in-frame deletion spanning the hinge region and a part of the hormone-binding domain. If expressed, these mutant ERs would lack hormone-binding capacity and would be undetected by the anti-ER antibodies currently in clinical use. Genetic instability, when associated with mutant ERs in subpopulations of breast tumor cells, may provide the selective pressure leading to hormone resistance. T47DCO cells and their clonal derivatives provide a model for the systematic study of ER mutations and other mechanisms of hormone resistance in Stage IV breast cancer.

Amino Acid Sequence↗

PubMed1990

TSH subunit gene promoters from a murine alpha-subunit producing tumor function normally.

The murine thyrotropic MGH101A tumor is characterized by absent thyrotropin (TSH) beta gene expression and altered thyroid hormone (T3) regulation of the alpha-subunit. Comparison of the promoter structures of both alpha and TSH beta subunit genes from MGH101A with the promoter in expressing TtT-97 thyrotropes revealed no detectable differences. Transfection of the TSH beta promoter from MGH101A linked to luciferase showed minimal expression in primary or cloned MGH101A cells, or L-cells. However, a 6- to 10-fold increase in expression was exhibited in transfected thyrotropes. For the alpha gene, promoter activity was highest in thyrotropes and in cloned MGH101A cells, 5-fold lower in MGH101A tumors, and 10-fold lower in L-cells. Both promoters were not substantially affected by T3 treatment in MGH101A cells. In thyrotropes, promoter activity was inhibited 62.5% and 57.7% by 10 nM T3 treatment for the TSH beta and alpha genes, respectively. DNase I protection showed that factors from TtT-97 but not from MGH101A cells interacted with regions in the TSH beta promoter, while nuclear extracts from each tumor demonstrated at least one protein-DNA interaction with the alpha-subunit promoter. These studies suggest that the molecular defects in the MGH101A tumor are related to the absence of trans-acting factors and are not a result of altered primary gene structure.

Animals↗

PubMed1990

Thyrotrope expression and thyroid hormone inhibition map to different regions of the mouse glycoprotein hormone alpha-subunit gene promoter.

The alpha-subunit gene of the glycoprotein hormones is normally expressed in pituitary thyrotropes and gonadotropes and in placental cells. Thus, this gene must contain elements that mediate expression and hormonal responses in different cell types. The localization of DNA regions important for expression and regulation of the alpha-subunit gene in thyrotrope cells has not previously been reported. In these studies luciferase expression constructs containing 1700 basepairs of 5' flanking DNA derived from the mouse alpha-subunit gene were introduced by electroporation into freshly dispersed cells from TSH-producing mouse pituitary tumors (TtT 97). This promoter functioned with greater efficiency in thyrotropes than in nonthyrotrope pituitary GH4 cells and L-cell fibroblasts. Primer extension confirmed that transcription from the alpha-subunit constructs initiated at the same site as the endogenous gene. Studies using 5' truncations showed a progressive loss of alpha-subunit promoter activity in thyrotropes between -480 and -120, with regions upstream of -254 contributing substantially to expression in thyrotrope cells. Thyroid hormone inhibited alpha-subunit promoter activity in a dose-dependent fashion, although in vivo treatment of tumors with thyroid hormone before transfection was necessary to achieve maximal inhibition. Thyroid hormone inhibition of alpha-subunit promoter activity also occurred in GH4 cells, but no effect was observed in L-cells. Studies using 5' truncations localized a region responsible for thyroid hormone inhibition between -62 and +43, encompassing the TATA sequence and the transcriptional initiation site. When this region was compared to the thyroid hormone inhibitory regions of the alpha-subunit genes from other species and the mouse TSH beta-subunit gene, a 6-basepair motif, 5' (G/A)GTG(G/A)G 3', emerged as a possible consensus sequence for a thyroid hormone inhibitory element.

Animals↗

PubMed1990

Activation of the murine thyrotropin beta-subunit promoter by GH4 rat pituitary cell-free extracts.

Expression of the TSH beta subunit gene is restricted to the thyrotroph cells of the anterior pituitary. Previously we identified several AT-rich DNA elements within the murine (m) TSH beta 5'-flanking region, denoted as D1 (-253 to -227), P4 (-142 to -131), P3 (-126 to -112), P2 (-106 to -98), and P1 (-76 to -68) which bind thyrotroph-specific factor(s). These sites are related to, but distinct from GHF-1 and LSF-1 binding sites, which restrict GH and PRL gene expression to pituitary somatotrophs and lactotrophs, respectively. To determine whether different pituitary cell types contain related factors capable of activating the mTSH beta promoter, cell-free transcription studies were performed using extracts from GH4 rat pituitary somatomammotroph cells. AI-through the endogenous mTSH beta gene is not expressed in GH4 cells, in vitro transcription of the mTSH beta promoter, normalized to the Rous sarcoma virus internal control, revealed faithful transcription initiation from the authentic mTSH beta CAP sites in GH4 but not in HeLa cell extracts. Cell-free transcription analysis of mTSH beta 5'-deletion mutants revealed consistent promoter activity with deletion to position -46 but complete loss of activity when deleted to position -9. To better define the specific factors in pituitary somatomammotrophs which interact with and activate the mTSH beta promoter, DNase I protection and gel-shift studies were performed using extracts from GC rat pituitary somatomammotroph cells and DNA affinity-purified lactotroph-specific transcription factor, LSF-1, required for rat PRL promoter activity, and purified from GC cells. These cells contain a factor(s) which binds to thyrotroph-specific elements of the mTSH beta promoter. These studies also show that LSF-1 binds the D1 and proximal thyrotroph-specific elements of the mTSH beta promoter and is capable of reconstituting the trans-activation of the mTSH beta promoter in HeLa nonpituitary cell extracts in vitro. Conversely, nuclear factors present in TtT-97 murine thyrotrophs bind the proximal lactotroph-specific elements on the rPRL promoter. This in vitro transcription assay provides a means to biochemically dissect the trans-activation of the mTSH beta promoter and to determine the functional overlap of distinct pituitary cell-specific factors in regulating GH, PRL, and TSH beta gene expression.

Animals↗

PubMed1990

Protein factors in thyrotropic tumor nuclear extracts bind to a region of the mouse thyrotropin beta-subunit promoter essential for expression in thyrotropes.

The beta-subunit gene of TSH is specifically expressed in thyrotrope cells of the anterior pituitary gland. To define the particular TSH beta-subunit gene sequences responsible for tissue-specific expression, TSH beta promoter fragments were assessed for promoter activity by gene transfer into TSH-expressing thyrotropic tumor cells (TtT-97). Previous studies have shown that the murine TSH beta gene promoter was more efficiently used in TtT-97 cells compared to other pituitary-derived cells or nonpituitary fibroblasts and that a 191-basepair DNA sequence of the 5' flanking region between -271 and -80 was sufficient for maximal promoter activity in thyrotropes. Further deletional analysis within this region has localized the area responsible for expression in thyrotropes to a 37-basepair region between -117 and -80 up-stream of the major transcriptional initiation site. DNase-I protection assays demonstrated that this functionally defined 5' flanking area, in addition to the adjacent sequences immediately up-stream and down-stream, interacts with protein factors present in nuclear extracts from TtT-97 tumor cells. When fused to a heterologous promoter, fragments derived from the region between -271 and -80 exhibited cell-specific activity, although this was not conferred solely by the TSH beta promoter fragment from -117 to -80. Heterologous promoter activity was further stimulated when fragments containing the areas from -271 or -201 to -77 were used, suggesting combinatorial cis interactions between these regions of the TSH beta promoter. DNase-I protection studies suggest that there are multiple protein-binding domains in the mouse TSH beta 5' flanking sequence. Only the more proximal domains, which encompass important promoter elements, appear to be required for efficient expression in thyrotropes, whereas other more up-stream sites of protein interaction may be involved in regulatory aspects of TSH beta gene expression.

Animals↗

PubMed1990

Identification of cis-acting promoter elements important for expression of the mouse glycoprotein hormone alpha-subunit gene in thyrotropes.

The glycoprotein hormone alpha-subunit gene is expressed in a cell-specific manner in the anterior pituitary and placenta. Previous studies have shown that the region between -178 to -111 is indispensable for placental-specific expression of the human alpha-subunit gene. Using gene transfer techniques with chimeric luciferase plasmids, this report identifies regions of the mouse alpha-subunit promoter that are important for transcriptional activation in primary thyrotropic cells. Transient expression of a series of 5' flanking DNA deletions resulted in stepwise reductions of basal promoter activity between -480 to -417 (4-fold), -254 to -177 (5-fold), and -177 to -120 (3.5-fold). DNase-I protection analysis with nuclear extracts from thyrotropic tumor cells revealed specific protein-DNA interactions within each of these functionally defined regions. These were mapped to positions -474 to -452, -447 to -419, -213 to -170, and -158 to -101 within the 5' flanking region. In contrast, in mouse fibroblast L-cells no significant difference in alpha-subunit promoter activity was found by deleting the region from -480 to -177. However, a 3-fold decrease, similar to that found in primary thyrotropes, was found by deleting the region from -177 to -120. Further, a smaller region between -138 and -122 was the only area detected by the DNase-I protection assay using L-cell nuclear extracts. Thus, several cis-acting promoter elements located up-stream of position -177 are important for expression in thyrotropes. These elements also bind nuclear factors present in thyrotropes but not in nonpituitary fibroblasts and, therefore, differ from those mediating expression of the human alpha-subunit gene in the placenta.

Animals↗

PubMed1990

Lipoprotein lipase gene expression in rat adipocytes is regulated by isoproterenol and insulin through different mechanisms.

Lipoprotein lipase (LPL) is highly regulated by catecholamines and insulin in adipocytes. Isoproterenol, a beta-adrenergic agonist, decreases LPL enzyme activity, whereas insulin increases LPL activity. We have isolated an 868-basepair rat LPL cDNA clone to assess hormone-mediated changes in LPL steady state mRNA levels and LPL gene transcription rates in adipocytes. Northern blot analysis of isoproterenol-treated (10(-6) M) adipocytes showed that LPL steady state mRNA decreased by 15 min. Nuclear run-on transcription assays in isoproterenol-treated cells indicated that LPL gene transcription was also decreased at 15 min compared to that in control cells. Conversely, insulin (6.7 x 10(-8) M) mediated an increase in LPL steady state mRNA in treated adipocytes, yet LPL gene transcription was not different from that in control cells. Thus, the isoproterenol-mediated decrease in LPL enzyme activity and steady state mRNA levels in adipocytes is associated with decreases in LPL gene transcription. Insulin, which does not affect LPL gene transcription, increases LPL enzyme activity and steady state mRNA levels. The effect of insulin on LPL mRNA is probably due to insulin-induced changes in mRNA stability.

Adipose Tissue↗

PubMed1990

Transcriptional regulation of the murine TSH subunit genes.

These studies have demonstrated that the murine TSH subunit genes are sensitively regulated by thyroid hormone. The T3 receptor complex interacts with both the TSH beta and alpha-subunit gene either in or near the 5' flanking region in close proximity to the transcriptional start sites. This interaction interrupts transcription from the start sites, thus decreasing transcription of the two genes. As a result steady state mRNA levels of both TSH beta and alpha-subunit genes are decreased in the cytoplasm of thyrotropic cells. This series of interactions explains most of the effects of T3 on TSH biosynthesis.

Animals↗

PubMed1989

Thyroid hormone regulates the mouse thyrotropin beta-subunit gene promoter in transfected primary thyrotropes.

In TtT 97 cells, a thyrotropin-producing mouse pituitary tumor, thyroid hormone rapidly inhibits the transcription rate of both the thyrotropin alpha- and beta-subunit (TSH beta) genes, and this closely parallels the increase in nuclear thyroid hormone receptor occupancy. In this study, we have identified regions of the mouse TSH beta gene which are involved in mediating tissue-specific and thyroid hormone-regulated expression. Transient expression studies were performed using a series of chimeric plasmids in which 5'-flanking DNA was ligated to the firefly luciferase gene. Following transfection by electroporation, efficient expression of TSH beta 5'-flanking luciferase constructs occurred only in cells derived from TtT 97 tumors which express the endogenous TSH beta gene. Deletion analysis demonstrated that the region of the 5'-flanking DNA between positions -271 and -80 relative to the major transcriptional start site is important for TSH beta promoter activity in thyrotropes. No expression was measurable in mouse L cells, a fibroblast line, whereas a low level of expression was seen in MGH 101A cells derived from a thyrotropic tumor which no longer expresses the TSH beta gene. Reduced expression of TSH beta constructs was also found in GH3 and GH4 pituitary tumor lines. Addition of thyroid hormone effectively inhibited the level of transient TSH beta promoter activity in TtT 97 cells in a dose-dependent manner. The inhibitory effect was more pronounced and more accurately reflected the transcription rate data when transfected cells were derived from tumors treated with thyroid hormone for 5 days prior to transfection. Deletion of all but 46 base pairs of TSH beta gene 5'-flanking DNA and 3 base pairs of the first exon had no effect on thyroid hormone inhibition. This indicates that signals sufficient for transcriptional regulation of the TSH beta gene by thyroid hormone reside in the vicinity of the proximal promoter and may act by interfering with basal transcriptional factors.

Animals↗

PubMed1989

IL-1 beta production in cultured human monocytes is regulated at multiple levels.

Cultured human monocytes and tissue macrophages exhibit a marked decrease in LPS-stimulated IL-1 production. Preincubation of monocytes in 100 U/ml IFN-gamma or in 0.25 microgram/ml cycloheximide (Cx) leads to a partial maintenance of the ability to produce IL-1 in response to subsequent stimulation with LPS, whereas preincubation in both reagents leads to a near complete maintenance of this response. These studies were performed to determine the mechanisms of these alterations in regulation of IL-1 production in cultured monocytes. In comparison to LPS-induced fresh monocytes, cells preincubated in medium for 1 day, then stimulated for 24 h with 100 ng/ml LPS, exhibited a 50% or more decrease in steady-state IL-1 beta mRNA levels. This reduction was accompanied by a four- to fivefold decrease in relative transcriptional rate, as determined by the nuclear run-on technique. The medium-aged cells also displayed greatly decreased IL-1 beta production with deficient secretion. Monocytes preincubated in IFN-gamma for 1 day before LPS stimulation exhibited a variable maintenance in IL-1 beta production, secondary both to prolonged duration of transcription and to increased IL-1 beta mRNA stability. Monocytes were preincubated for 1 day in Cx alone or in both IFN-gamma and cycloheximide, then these substances were washed out before a subsequent 24-h culture in LPS. Cx preincubation led to increases in both the peak and duration of transcription as well as to enhanced secretion of IL-1 beta protein. Monocytes preincubated in both IFN-gamma and Cx exhibited increased IL-1 beta mRNA levels due to both enhanced transcription and mRNA stability; more IL-1 beta protein secretion also was present in these cells. In summary, IFN-gamma and Cx may maintain LPS-induced IL-1 beta production in cultured human monocytes through multiple mechanisms. These alterations in regulation of IL-1 beta production during monocyte differentiation may be relevant to tissue macrophages where impaired IL-1 production may be present.

Blood Proteins↗

PubMed1989

Clinical and molecular studies of a thyrotropin-secreting pituitary adenoma.

A 40-year-old woman, who had previously received radioactive iodine for hyperthyroidism, presumably due to Graves' disease, subsequently was found to have inappropriately elevated serum TSH and alpha-subunit levels and a pituitary adenoma. Detailed clinical studies revealed marked serum TSH elevations (approximately 100 mU/L) with no circadian variation, but with 7 pulses/24 h. Serum alpha-subunit levels averaged 2.5 micrograms/L, with 13 pulses/24 h. Neither serum TSH nor alpha-subunit responded to TRH stimulation, nor did serum TSH change during dopamine infusion, but alpha-subunit levels did decline slightly. In contrast, during somatostatin infusion, serum TSH declined to 30% of baseline levels, while alpha-subunit levels did not change. Pituitary adenoma tissue obtained at the time of transsphenoidal surgery immunostained weakly with anti-TSH beta serum and strongly with anti-alpha-subunit serum. Northern blot analysis of RNA isolated from the tumor revealed TSH beta and alpha-subunit mRNA levels of normal length, while primer extension analysis showed a major initiation site for the TSH beta gene that appeared to be identical in the tumor and normal pituitary tissue. A second minor upstream start site was detected in the tumor, but it represented less than 1% of transcription compared to the major downstream start site. We conclude that the tumor secreted TSH and alpha-subunit in an abnormal and discordant fashion, but that the TSH gene initiation site appeared to be normal and, therefore, did not explain the observed secretory abnormalities.

Adenoma↗

PubMed1989

Identification of thyrotroph-specific factors and cis-acting sequences of the murine thyrotropin beta subunit gene.

Pituitary thyrotroph cells specialize in the synthesis of TSH, and thus represent a model to study cell-specific gene expression. We have used the murine TSH beta (mTSH beta) gene promoter and TSH-producing and nonproducing transplantable tumors derived from murine thyrotroph cells, referred to as TtT-97 and MGH 101A, respectively, to identify nuclear factors which selectively interact with the mTSH beta gene. DNase I protection analyses demonstrate that factors present in TtT-97 nuclear extracts bind with high affinity to five separate sites in the TSH beta promoter region, denoted as distal D1 (-253 to -227) and proximal, P1 (-76 to -68), P2 (-106 to -98), P3 (-126 to -112), and P4 (-142 to -131) footprints. By contrast, non-TSH beta expressing thyrotroph cell nuclear extracts and L-cell nonpituitary cell extracts did not appear to footprint the D1 site; whereas the nonpituitary nuclear extracts revealed minimal DNase I protection in the P1-P4 regions. These data show that the distal D1 site is thyrotroph specific and contains a 6 base pair direct repeat sequence (5'-AGATAT-3'). Factor occupancy of the D1 site is protein dependent, occurs rapidly (less than 15 sec), is destabilized by 170 mM KCl, and results in an associated DNase I hypersensitive region. A double-stranded oligonucleotide spanning the D1 footprint competes only the distal factor binding region. Transfection of plasmid constructs containing progressive 5'-deletions of the mTSH beta promoter linked to the reporter gene luciferase into primary TtT-97 cells demonstrate a marked decrease in activity between the regions -270 and -79, which contains the D1 region.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1988

Human progesterone A-receptors can be synthesized intracellularly and are biologically functional.

In order to investigate the origin and functional independence of the human progesterone receptor A binding protein, we have expressed a truncated human progesterone receptor cDNA in both gene transfer and in vitro translation assays. Proteins identical in size and antigenicity to the A-receptors found naturally in human progesterone target cells are synthesized from this cDNA that lacks the putative B receptor initiator methionine codon of the complete cDNA. The functional independence of A-receptors is suggested by their ability to bind hormone and to stimulate transcription from the progestin responsive mouse mammary tumor virus promoter.

Blotting, Western↗