Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:2.7.1.21 (thymidine kinase)
7,561 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The dihydropyridine Ca2+ channel modulators (-) Bay K 8644 (R5417) and nimodipine were used to study the role of voltage-gated Ca2+ channels in the regulation of PRL gene transcription in GH3 cells. Fusion constructs containing 5'-flanking sequences from the rat PRL gene linked to either the bacterial chloramphenicol acetyltransferase (CAT) gene or the firefly luciferase gene were transiently expressed in GH3 cells and the transcriptional response to Ca2+ channel modulators was assessed. The Ca2+ channel agonist R5417 enhanced the transcription of a PRL-CAT fusion gene containing 2.5 kilobase (kb) pairs of the 5'-flanking sequence. This response was completely blocked by the Ca2+ channel blocker nimodipine demonstrating that sequences in the PRL 5'-flanking region confer response to Ca2+. Transfection with PRL-CAT constructs containing 2.5 kb to 0.6 kb pairs of 5'-flanking sequence were responsive to Ca2+, although those which contained the distal enhancer region (positions-1765 to -1495) had much higher basal expression. The possibility that the distal enhancer might contain Ca2(+)-responsive elements was tested by comparing the response to R5417 and TRH for both the proximal enhancer region (approximately first 300 base pair of the 5'-flanking sequence) and distal enhancer regions linked to the thymidine kinase promoter and CAT. The results demonstrate that these two regions contribute to the overall transcriptional response to Ca2+ and TRH. The distal region does not confer a response to phorbol ester, while the proximal region is responsive to that treatment.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Pituitary calcium channel modulation and regulation of prolactin gene expression. 170 75

Two TRH-responsive elements have been identified in the rat TSH beta gene by deletion/mutation analysis of the 5'-flanking region of the gene and transfection of TSH beta-luciferase constructs into the GH3 pituitary cell line. Biological responsiveness was confirmed by inserting synthetic oligonucleotides next to the heterologous viral thymidine kinase (tk) promoter in tk luciferase (tkLUC) constructs. Both DNA regions, termed TSH A (at -274 to -258 bp) and TSH C (-402 to -385 bp), have a high level of sequence similarity to binding sites for the POU domain pituitary transcription factor Pit-1/GHF-1. In transfection assays, the TSH A region had no basal enhancer activity, but did confer 3- to 6-fold TRH- and PMA-stimulated transcriptional responses to the tk promoter. The TSH C region conferred basal enhancer activity (3- to 10-fold above control tkLUC) as well as a 2- to 3-fold TRH or PMA response. Combinations of TSH A and TSH C elements conferred both enhancer activity and a TRH- or PMA-stimulated response, but more than two copies of the regions resulted in no further stimulatory effect. Both TSH beta gene regions bound to nuclear proteins from GH3 cells, as determined by gel retardation analysis. The TSH A region DNA formed three prominent DNA-protein complexes, ranging from slowly to rapidly migrating bands and with calculated affinities of 32, 0.5, and 208 nM, respectively. The TSH C region formed two major complexes, which corresponded on the basis of mobility to the most slowly and rapidly migrating complexes formed by TSH A, but with calculated affinities of 3.1 and 33 nM. TSH C also formed a rapidly migrating minor complex unique for this gene region. The more rapidly migrating complexes appeared to be specific to nuclear proteins from GH3 cells. Treatment of cells with TRH did not significantly alter the affinity of protein binding. Mutation of TSH A and TSH C DNA by T to G substitutions abolished the ability of the DNA to confer a TRH response and severely inhibited the ability of the DNA to bind to GH3 nuclear proteins. Thus, transcriptional regulation of the rat TSH beta gene by TRH is correlated with the ability of the two TRH-sensitive elements to bind nuclear proteins. The differences noted in basal enhancer activity or the degree of TRH responsiveness may be related to some unique proteins bound to each DNA or to the differences in affinity of binding of the proteins common to both elements.
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PMID:Thyrotropin (TSH)-releasing hormone-responsive elements in the rat TSH beta gene have distinct biological and nuclear protein-binding properties. 173 70

Our previous studies demonstrated TRH stimulation of TSH beta gene transcription in rat pituitary cell cultures and in transient expression assays, with the TRH-sensitive region located between -1.3 kilobases and -204 basepairs (bp) relative to the major transcriptional start site. Using nuclear runoff and transient expression assays, we have analyzed the interactions among TRH, the phorbol ester 12-myristate 13-acetate (PMA), and the adenylate cyclase activator forskolin on TSH beta gene transcription. In cultured pituitary cells, TSH beta gene transcription was stimulated by 2 h of 10(-9) M TRH (2- to 4-fold), 100 nM PMA (2- to 6-fold), or 2 microM forskolin (1.5- to 2.5-fold) treatment, with additive interactions among all three effectors. Chimeric plasmids containing various 5'-flanking portions of the TSH beta gene and both transcriptional start sites, fused to the chloramphenicol acetyltransferase (CAT) gene, were transfected into the clonal pituitary GH3 cell line to delineate DNA sequences conferring this regulation. Transfected TSH beta CAT constructs containing TSH beta gene sequences from -2100/+27I150, -1295/+27I150, and -520/+27I150 expressed CAT enzyme activity which was stimulated by 24 h of TRH (2- to 3-fold), PMA (3- to 6-fold), or forskolin (1.5- to 3-fold) treatment, similar to observations in normal pituitary cells. In addition, a CAT expression vector construct containing only upstream TSH beta gene sequences from -703 to -85 bp, fused to the heterologous thymidine kinase promoter (tkCAT), exhibited similarly stimulated transcription in a transfection assay in response to TRH, PMA, and forskolin.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Interactions of thyrotropin-releasing hormone, phorbol ester, and forskolin-sensitive regions of the rat thyrotropin-beta gene. 217 92

In order to investigate the molecular mechanism(s) by which TRH regulates the biosynthesis of TSH, we are studying the effects of TRH on the expression of the TSH subunit genes (alpha and TSH beta). To study the structure-function relation of TRH stimulation of the activity of the single rat TSH beta gene, chimaeric plasmids were constructed. The 5'-flanking region of the rat TSH beta gene including exon 1 (5'-untranslated region) was inserted into a promoterless, modified pBR, chloramphenicol acetyltransferase (CAT) expression vector. After transfection, specific TSH beta promoter activity was evident in both TRH-responsive pituitary-derived GH3 and primary pituitary cell cultures. To determine potential regulation of TSH beta promoter-directed activity in these cells by TRH, cells were incubated with media containing TRH (10(-7) to 10(-11) M) for 1 to 48 h. TRH stimulated a 1.5- to 3-fold increase in TSH beta promoter activity. Concomitant with an increase in CAT activity was an anticipated increase in PRL synthesis in the GH3 cells in response to TRH. The TRH effect on the TSH beta gene was specific; no increase in CAT activity was detected for TKCAT (thymidine kinase of herpes simplex virus promoter), pBRCAT (no promoter), or TSH beta CAT (3'-5'-orientation). Similar results were obtained using primary pituitary cell cultures. Deletion mutation analysis indicated that TRH sensitivity was detected in a 1.1 kilobase, but not in a 0.38 kilobase TSH beta gene fragment suggesting that the TRH responsive element(s) resides at least in part within the 700 base pairs of the 5'-flanking sequence.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Thyrotropin-releasing hormone stimulates the activity of the rat thyrotropin beta-subunit gene promoter transfected into pituitary cells. 249 52

The ability of an upstream element of the rat PRL gene to permit transcriptional regulation in response to several different hormones has been examined. To test the ability of specific DNA sequences to mediate hormone responsiveness, DNA fragments were subcloned upstream of a thymidine kinase-chloramphenicol acetyltransferase fusion gene and transferred into GH3 pituitary tumor cells. Initially, fragments representing a distal enhancer element (positions -1713 to -1495) and a more proximal element (positions -292 to -38) were tested. The results demonstrate that the distal enhancer permits cAMP, TRH, epidermal growth factor (EGF), and estradiol to stimulate expression of the thymidine kinase-chloramphenicol acetyltransferase gene. The proximal element permitted fusion gene regulation in response to cAMP, TRH, EGF, and phorbol esters. For the cAMP, TRH, and EGF responses, the distal element permitted responses approximately equal to or greater than responses conferred by the proximal PRL gene fragment. The response of the distal element to cAMP and TRH was more than additive with the response to estradiol, suggesting that the estrogen response element is distinct but may interact cooperatively with the other hormone response elements. Mutation of the estrogen-responsive element abolished both the response to estrogen and the cooperative response with cAMP, but not the response to cAMP itself. Mutation of a sequence involved in basal enhancer activity of the distal element reduced both basal transcription and the response to cAMP. These results suggest that the distal enhancer sequence of the PRL gene contains, in addition to an estrogen response element, elements that confer responsiveness to cAMP, TRH, and EGF.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:The distal enhancer region of the rat prolactin gene contains elements conferring response to multiple hormones. 253 91

Human PRL (hPRL)-secreting adenoma cells obtained at hypophysectomy were fused with a mutant mouse fibroblast line (LMTK-) which is aminopterin sensitive due to a deficiency in the enzyme thymidine kinase. After fusion with polyethylene glycol, cells containing nuclear material from the two parental lines (heterokaryons) were selected in medium containing hypoxanthine, aminopterin, and thymidine, and resultant clones were screened for hPRL secretion. Functional human X mouse somatic cell hybrid clones secreting hPRL were isolated in order to study hPRL gene expression and regulation. Positive hybrid clones were subcultured and have sustained hPRL secretion. The hybrid nature of the cells was confirmed by fibroblastic morphology resembling the mouse parental cell, mixed karyotype of mouse and human chromosomes, and mixed isozyme banding pattern for human and mouse glucose-6-phosphate dehydrogenase and malic enzyme. Specific expression of the hPRL gene was demonstrated by the presence of electron microscopic secretory granules (650-800 nm), positive immunoperoxidase staining using anti-hPRL serum, and sustained secretion of immunoreactive hPRL, which comigrated with [125I] hPRL standard on Sephadex chromatography. Hormonal modulation of hPRL gene expression by TRH was dominantly expressed in the hybrid cell. Human chromosome 6 was identified in hybrid cells secreting hPRL, and the cells expressed human malic enzyme, a marker for this chromosome, thus confirming the chromosome assignment of the hPRL gene. The results show that functional replicating hybrids secreting hPRL can be isolated. The technique provides a useful in vitro model for the study of hPRL gene expression and modulation.
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PMID:Isolation of human-mouse somatic cell hybrids producing human prolactin: dominant expression of hormone secretion and regulation. 640 19

TRH is known to stimulate the transcription of the TSH gene in pituitary cells. To examine TRH-responsive elements of the human TSH alpha-subunit gene, we have used transient transfection of GH3 rat pituitary tumor cells. Using this system, TRH treatment stimulated expression of a reporter gene containing 846 base pairs from the 5'-flanking region of the human glycoprotein hormone alpha-subunit gene linked to luciferase. Analysis of 5'-deletions of the alpha-subunit sequence revealed that at least two DNA regions with upstream limits between positions -223 to -190 and positions -151 to -135 are important for regulation by TRH. The more proximal region includes a previously defined cAMP-response element (CRE) while the more upstream region contains an element with sequence similarity to the binding site for the pituitary transcription factor, Pit-1. The TRH responsiveness of each individual region was tested by inserting fragments upstream of a thymidine kinase-luciferase reporter gene. The -151 to -100 region had basal enhancer activity and permitted a 3.4-fold response to TRH. The -223 to -168 region did not permit a TRH response, but possessed basal enhancer activity. The combination of both regions resulted in a 5-fold stimulation by TRH. To assess the contributions of different signal transduction pathways, various combinations of treatments were examined. Combined treatment with TRH and forskolin led to an additive activity. Treatment with TRH plus phorbol 12-myristate-13-acetate resulted in the same level of reporter gene activity as with either agent alone.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Involvement of a cAMP-responsive DNA element in mediating TRH responsiveness of the human thyrotropin alpha-subunit gene. 751 24

Three regions within the 5'-flanking region of the TSH beta gene have A-T-rich sequences which have sequence similarity to binding sites for the pituitary-specific POU domain transcription factor Pit-1/GHF-1. These three regions have been termed TSH A (-274 to -258 bp), TSH B (-336 to -326 bp), and TSH C (-402 to -384 bp). TSH A and TSH C are able to confer 2-6-fold TRH stimulation to the heterologous viral thymidine kinase (tk) promoter in transient expression assays in GH3 pituitary cells; TSH C can confer a 3-10-fold increase in basal enhancer activity as well. TSH A, B, and C DNAs all bound Pit-1 from GH3 cell nuclear extracts, based on gel mobility shift analysis in which antibody against Pit-1 prevented the formation of specific DNA-GH3 nuclear protein complexes. TSH A and TSH C also each formed several additional DNA-nuclear protein complexes which were not observed with TSH B. Some of these complexes may contain Pit-1 as their formation was inhibited by the addition of Pit-1 antibody; other complexes, however, were not altered by antibody treatment. All three A-T-rich elements bound in vitro translated Pit-1, with calculated affinities of 360 (A), 125 (B), and 38 (C) nM, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Pit-1/GHF-1 binds to TRH-sensitive regions of the rat thyrotropin beta gene. 836 38

Retinoid X receptors (RXRs) form heterodimers with thyroid hormone receptors (TRs). RXRs increase DNA binding affinity of TRs and T3-mediated transactivation on positive T3 response elements (TREs). However, the role of RXRs on negative TREs, and the relation of RXRs to the dominant negative effect of mutant TRs, are not defined. To clarify the function of RXRs on negative TREs, we performed transient cotransfection studies using the rat glycoprotein hormone alpha promoter fused to luciferase gene (alphaLuc), and human TRH promoter fused to luciferase gene (TRH-Luc) as reporters. We found that the JEG-3 cell-alphaLuc system was very sensitive to TR regulation. Using TRbeta1 wild-type (WT) expression vector, 6.2 ng/well (170 ng/10 cm dish), and 0.2 ng/well (11 ng/10 cm dish) caused maximal, and half maximal, inhibition of Luc activities in the presence of 1 nM T3. A T3 dose dependent inhibition study was also performed. From these studies, we determined that the appropriate conditions in which to study alphaLuc transactivation, in a linear portion of the dose response curve, was using 0.8 ng/well TRbeta1 expression vector and 0.1 nM T3. Under these conditions, TRbeta1 mutant R316H (GH), but not G345R (Mf), showed a weak dominant negative effect at a 1:1 ratio in the presence of 0.1 nM T3 although neither mutant had detectable T3 binding affinity. Moreover this dominant negative effect of R316H on the alphaLuc reporter was enhanced in the presence of RXRgamma. Mutant G345R showed a stronger dominant negative effect than did R316H when using a double palindromic TRE fused to herpes simplex thymidine kinase-Luc reporter as a positive TRE. These results conform to the clinical features of R316H which is associated with apparent pituitary resistance of thyroid hormone (PRTH). Mutant R316H also showed a weak dominant negative effect with TRH-Luc at a 1:1 ratio in the absence or presence of RXRgamma. However RXRgamma did not enhance the dominant negative effect as it did using alphaLuc reporter gene. Electrophoretic gel mobility shift assay (EMSA) showed that RXR alpha augmented the DNA binding affinity of wild type and R316H TRs as heterodimers on the previously reported negative TREs of glycoprotein hormone alpha promoter, suggesting that RXR does not produce its response by removing TRs from these TREs. RXR alpha augmented DNA binding affinity of TRbeta1WT, and R316H showed a weaker heterodimer band than did the wild type in EMSA. Using the TRH-Luc reporter, basal activity was increased by wild type TRbeta1. However a TRbeta1 DNA binding domain mutant, (C127S) which can not bind to DNA, did not increase the basal activity. This indicates that DNA binding of the TR is required for increasing basal activity of TRH promoter. These results indicate that (1) RXR-TR heterodimers play a role in basal transactivation and T3 suppression of negatively regulated genes, and (2) RXRs increase the dominant negative effect of some mutant TRs on specific negative TREs. (3) This effect occurs without removing TRs from the TRE. (4) The differential dominant negative effect of mutant R316H (negative TRE > positive TRE) may explain, at least in part, the presentation of R316H as PRTH. (5) Augmentation of basal activity by wild type TRs on a negative TRE requires DNA binding.
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PMID:The function of retinoid X receptors on negative thyroid hormone response elements. 914 79

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