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)

Developmental control of gene expression often results from the coupling of growth arrest with the establishment of differentiation programs. QR1 is a gene specifically expressed in retinas during the late phase of embryogenesis. At this stage neuroectodermal precursors have reached terminal mitosis and are undergoing differentiation into distinct cell types. Transcription of the QR1 gene is tightly regulated during retinal development: this gene is expressed between embryonic day 9 (ED9) and ED17 and is completely repressed at hatching in quail. Moreover, QR1 transcription is downregulated when postmitotic neural retina cells are induced to proliferate by pp60v-src. We studied the stage-dependent transcriptional control of this gene during quail neural retina (QNR) cell development. Transient transfection experiments with QR1/CAT constructs at various stages of development showed that a region located between -935 and -1265 bp upstream of the transcription start site is necessary to promote transcription in retina cells during the late phase of embryonal development (QNR9, corresponding to ED9). By in vivo footprinting assays we identified at least two elements that are occupied by DNA-protein complexes in QNR cells: the A and B boxes. The A box allows formation of several biochemically distinct complexes: C1, C2, C3, and C4. Formation of the C2 complex mainly during early stages (ED7) and of C2, C3, and C4 complexes during postnatal life correlates with repression of QR1 transcription, whereas the C1 complex is strongly induced at ED11 when the QR1 gene is expressed. We previously showed that C1 was involved in downregulation of QR1 transcription by pp60v-src. Several complexes are also formed on the B box. We show that these complexes are exclusively present in neural tissues and that they involve members of the POU family of transcription factors. Mutations of each one of the two regions which abolish the binding of the C1 factor(s) on the A box and of the POU factor(s) on the B box also prevent stimulation of QR1 transcription in QNR9. Therefore, both elements appear to be required for the stage-specific transcription of the QR1 gene. We also show that the regulatory region from position -1265 to position -935 is able to confer stage-specific transcription upon a heterologous promoter (thymidine kinase). Indeed, this region stimulates transcription in differentiating retinas (QNR9) and represses transcription in terminally differentiated retinas (QNR17, corresponding to postnatal life). Our results suggest that cell growth regulation and developmental control are coordinated through the A and B boxes in regulating QR1 transcription during retinal differentiation.
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PMID:Developmental control of transcription of a retina-specific gene, QR1, during differentiation: involvement of factors from the POU family. 782 33

HC-11 mouse mammary epithelial cells stably transfected with a glucocorticoid-inducible Ha-ras construct encoding a transforming (val12) p21Ha-ras were cotransfected with a c-fos-CAT construct containing the human c-fos promoter up to position -711 and the CAT reporter gene. Expression of Ha-ras by dexamethasone leads to a transcriptional activation of the fos-CAT construct which was found to be sensitive to the PKC inhibitor ilmofosine (BM41440) and abrogated by PKC depletion following long-term exposure to TPA. The responsiveness to Ha-ras is retained if only the portion of the fos promoter covering the serum response element (SRE) and the adjacent fos AP-1 (FAP) site are put in front of a CAT gene linked to a thymidine kinase (TK) promoter. Further depletion of the FAP-site does not affect the inducibility by Ha-ras. Transcriptional activation of the SRE-FAP-TK-CAT as well as the SRE-TK-CAT constructs by Ha-ras is sensitive to the PKC-inhibitor ilmofosine (BM41440) and blocked by long-term exposure to TPA. Long-term exposure to TPA depletes cells of PKC alpha and significantly reduces the PKC epsilon levels. Long-term exposure in bryostatin 1 selectively depletes PKC alpha. Depletion of PKC alpha by bryostatin 1 does not reduce the transcriptional activation of the SRE-FAP-TK-CAT-construct by Ha-ras. It is concluded that (i) transforming Ha-ras induces c-fos in HC-11 cells via PKC (presumably epsilon), (ii) the signal is mediated to the serum response element (SRE) of the fos promoter and (iii) the fos AP-1 (FAP) site is not required for this mechanism.
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PMID:Role of protein kinase C in ras-mediated fos-expression. 794 78

Human papillomavirus type 16 (HPV16) induces squamous intraepithelial lesions of the cervical mucosa which may develop into invasive cancer. The expression of viral oncogenes in advanced neoplasias appears increased relative to the proliferating cell layers of low grade lesions raising questions about molecular mechanisms of deregulation of transcription. In a lymph node metastasis of a cervical cancer, we observed full-length HPV16 plasmids and molecules with a small deletion, which was mapped to the long control region (LCR). Both wild type and shortened LCR were amplified by PCR, cloned into the promoter test plasmid pBLCAT6 and sequenced to identify a 107 bp deletion from position 7794 to 7901 in the short LCR. CAT expression in cervical cancer-derived HT3, SiHa and CaSki cells appeared 5- to 6-fold increased under the control of the short LCR. This could be traced back to elevated levels of mRNA initiated at the viral oncogene promoter. A slight further increase in CAT expression was noted in the presence of the HPV16 E2 protein which is probably due to the deletion of one E2 binding site and consequent relief from E2 repression. Computer-assisted sequence analysis and band-shift experiments with purified YY1 protein and wild type or mutated oligonucleotides identified four binding sites for this cellular transcriptional repressor within the promoter-proximal segment of the HPV16 LCR, three of which were removed by the deletion. A LCR fragment comprising these YY1 binding sites was cloned in front of the heterologous thymidine kinase gene promoter and suppressed CAT expression 3- to 4-fold.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:The E6/E7 promoter of extrachromosomal HPV16 DNA in cervical cancers escapes from cellular repression by mutation of target sequences for YY1. 813 27

Expression of the mouse heat stable antigen (HSA or mouse CD24) shows tissue-specific as well as developmental regulation. During the maturation of several hematopoietic lineages, HSA expression is generally high in immature precursor cells and low or absent in terminally differentiated cells. We present evidence suggesting that this regulation of the HSA gene (Cd24a) occurs at the transcriptional level. In addition, sequence and methylation analysis of the Cd24a promoter revealed characteristics of both "housekeeping" and tissue-specific promoters, including a methylation-free, HpaII tiny fragment (HTF) island, multiple putative SP1 and AP-2 consensus binding sites, and a TATA box. Functional analysis of a 0.6-kilobase DNA fragment containing these elements fused to the CAT reporter gene in transient transfection experiments showed activity in both HSA expressing and non-expressing cell lines with a strength similar to that of the herpes-simplex virus-thymidine kinase promoter. Large fragments from the flanking region of the Cd24a promoter did not influence the ubiquitous nature of this promoter. Finally, we mapped the Cd24a, Cd24b, and Cd24c genes to mouse chromosomes 10, 8, and 14 respectively.
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PMID:The heat stable antigen (mouse CD24) gene is differentially regulated but has a housekeeping promoter. 822 59

Although G protein alpha subunits are known to regulate such cellular functions as growth and enzymatic activity, the ability of these proteins to regulate target gene expression has not yet been directly investigated. Transient expression in GH3 pituitary cells of a target rat prolactin promoter-chloramphenicol acetyltransferase construct, (-1957)PRL-CAT, was increased by coexpressed constitutively active alpha s mutant Q227L-alpha s but not by wild-type alpha s. Thus activated alpha s but not basal state alpha s can stimulate prolactin promoter activity. Q227L-alpha s also stimulated expression of construct (-187)PRL-CAT, showing that only the proximal prolactin promoter region is required for a response to activated alpha s. The promoter specificity of the transcriptional influence of activated alpha s was demonstrated by the inability of either Q227L-alpha s or wild-type alpha s to stimulate expression of control target constructs containing either the rat growth hormone promoter or the thymidine kinase promoter. Previous studies have shown that the most proximal prolactin promoter binding site for the pituitary-specific transcription factor pit-1, site 1P, can act as an independent response element for either thyrotropin-releasing hormone or Ca2+. Two copies of site 1P conferred upon a heterologous metallothionein promoter a response to Q227L-alpha s. This implies that site 1P can also serve as an independent response element for alpha s and suggests that pit-1 may be a mediator of the cellular regulation by alpha s of the prolactin promoter.
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PMID:Expression of constitutively active Gs alpha-subunits in GH3 pituitary cells stimulates prolactin promoter activity. 827 15

To define the molecular mechanisms involved in the action of gamma interferon (IFN-gamma), we have analyzed the transcriptional regulation of the mig (monokine induced by gamma interferon) gene, a member of the platelet factor 4-interleukin-8 cytokine family that is expressed in murine macrophages specifically in response to IFN-gamma. Analysis of mig/CAT chimeric constructs transiently transfected into the RAW 264.7 mouse monocytic cell line revealed a unique IFN-gamma-responsive element (gamma RE-1). The sequence of this cis regulatory element defined by deletion analysis contains an imperfect inverted repeat extending 27 bp. Examination of mig/CAT constructs with mutations in gamma RE-1 revealed that the palindromic positions in the element were essential for activity. Consistent with its function as an enhancer, a single copy of gamma RE-1 conferred IFN-gamma inducibility to a heterologous (herpes simplex virus thymidine kinase) promoter. Exonuclease III protection assays demonstrated symmetrical protection of a mig promoter fragment centered about the gamma RE-1 palindromic sequence. Using the gel electrophoretic mobility shift assay, we identified a factor (gamma RF-1) present in nuclear extracts prepared from IFN-gamma-stimulated RAW 264.7 cells which binds to gamma RE-1. The activation of gamma RF-1 occurred rapidly (within 1 min) in response to IFN-gamma and was independent of protein synthesis. Similar to the expression of mig mRNA, the formation of gamma RF-1 was selectively induced by IFN-gamma and not IFN-alpha. The regulation of gene expression through gamma RF-1 and gamma RE-1 may explain the preferential activation of a subset of interferon-inducible genes by IFN-gamma.
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PMID:A unique palindromic element mediates gamma interferon induction of mig gene expression. 828 31

Proopiomelanocortin (POMC) is expressed predominantly in the corticotrophic cells of the pituitary. Regulatory sequences required for the expression of the human (h) POMC gene were investigated using transient expression of hPOMC-CAT fusion genes in pituitary and nonpituitary cells in combination with DNase I footprint and gel retardation assays. Gene transfer experiments revealed that the hPOMC promoter is more efficiently transcribed in AtT-20 pituitary cells than in HeLa cells. Using deletion analysis, negative regulatory elements between nucleotides -676 and -414 and positive regulatory elements between nucleotides -414 and -93 could be identified. When placed in front of the heterologous thymidine kinase (tk) promoter, nucleotides -414/-223 enhance transcription in AtT-20 cells and in primary cultures of human pituitary tumor cells, but not in various nonpituitary cell lines. In contrast, a -112/-93 element enhances transcription of the tk promoter in all cells tested. DNase I footprint analysis revealed five sites protected by nuclear extracts obtained from AtT-20 cells within nucleotides -414 and -83 of the hPOMC promoter region. In contrast, only one of these sites (between nucleotides -115 and -83) was protected by nuclear extracts from HeLa cells. Gel mobility-shift experiments revealed that an oligonucleotide comprising nucleotides -112/-93 binds a novel nuclear protein. This protein may contribute to the non-cell type-specific expression of the hPOMC gene outside the pituitary, whereas at least five transcription factors seem to be required for high basal transcription of the gene in corticotrophic cells.
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PMID:Regulatory elements of the human proopiomelanocortin gene promoter. 832 20

These experiments were designed to examine the effect of structural modifications to the estradiol-17 beta (E2) molecule on the estrogen response element (ERE) dependent activation of the thymidine kinase (tk) promoter. Estrogen receptor (ER) positive MCF-7 cells were transfected with plasmids containing one or two vitellogenin EREs inserted upstream of the tk promoter in p(-37)tk. Transient expression of the CAT gene in these constructs was measured after cells had been maintained for 36-42 h in the presence of E2 or an E2 analogue. E2 induced CAT expression at levels as low as 10(-13) M, with maximum induction at 10(-11) M. CAT activity decreased at higher concentrations of E2. Estratriene, which has low affinity for ER, was active only at micromolar concentrations. 3-Hydroxyestratriene displayed maximal activity at 10(-9) M, with higher levels being less active. Still higher concentrations (10(-7) M) of estratrien-17 beta-ol were required to induce maximum CAT activity. All positional and conformational alterations in the D-ring hydroxyl group of E2 yielded active ligands. Movement of the phenolic hydroxyl group of E2 to other positions on the A-ring produced dihydroxyestrogens with varied capacities to activate CAT (2-hydroxyestratrien-17 beta-ol produced maximum CAT activation at 10(-11) M; 1-hydroxyestratrien-17 beta-ol required a 10(-8) M concentration for maximum activity; 4-hydroxyestratrien-17 beta-ol gave maximum CAT activation at 10(-6) M). Only those androstanediols or 5-androstenediols with a 3 beta-hydroxyl group were capable of activating CAT expression.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Effects of estradiol-17 beta analogues on activation of estrogen response element regulated chloramphenicol acetyltransferase expression. 833 31

p53 is known to bind specifically to the 44-bp human DNA sequence in an immunoprecipitation assay. We show here that the transcription of the reporter CAT gene linked with the herpesvirus thymidine kinase (tk) promoter containing the 44-base sequence is enhanced by mouse wild-type but not mutant-type p53 in F9 and p53-null Saos-2 cells. The p53-mediated transactivation was dramatically abrogated by introduction of SV40 large T antigen (SVLT) in Saos-2 cells in which p53 was clearly associated with SVLT. Furthermore, the p53-SVLT complex did not bind to the 44-base sequence at all. Thus, SVLT sequesters the transactivation function of the wild-type p53 by inhibiting the binding of p53 to the 44-base sequence. This is good evidence to show 'loss of functions' in the product of a tumor-suppressor oncogene by a dominant oncogene product at a molecular level.
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PMID:Abrogation of p53-mediated transactivation by SV40 large T antigen. 838 54

Glucocorticoids regulate the proliferation of mouse L cells. Incorporation of [3H]thymidine is inhibited by 70-90% within 24 h after addition of 0.1 microM dexamethasone. This effect on L cells is completely reversible. The expression of the thymidine kinase gene (Tk-1) has been examined in L cells that have been treated with 0.1 microM dexamethasone for 24 h. Dexamethasone inhibits thymidine kinase activity 70-90% after 24 h. This is associated with a 90-95% decrease in Tk mRNA abundance. The decrease in Tk mRNA is not caused by a decrease in transcription of Tk-1, as shown by nuclear run-on transcription assays. Transient expression of the CAT (chloramphenicol acetyltransferase) gene, driven by the Tk-1 promoter, was not affected by dexamethasone, and transcription of stably integrated Tk minigenes in LMTk- cells was not affected by dexamethasone. This effect was observed regardless of whether Tk cDNA was fused to the simian virus 40 promoter or the mouse Tk-1 promoter region. Conversely, expression of thymidine kinase was inhibited when stable Tk+ transformants of LMTk- cells were exposed to glucocorticoids; and inhibition of expression was observed irrespective of the promoter that was used to drive transcription of the Tk minigenes. These data indicate that glucocorticoid regulation of Tk-1 in mouse L cells is, within the limits of detection of the assays used in these studies, entirely due to a posttranscriptional mechanism.
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PMID:Glucocorticoid regulation of thymidine kinase (Tk-1) expression in L929 cells. 845 47


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