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 X. laevis tRNA((Ser)Sec) gene is different from the other tRNA genes in that its promoter contains two external elements, a PSE and a TATA box functionally equivalent to those of the U6 snRNA gene. Of the two internal promoters governing classical tRNA gene transcription, only subsists the internal B box. In this report, we show that the tRNA((Ser)Sec) contains in addition an activator element (AE) which we have mapped by extensive mutagenesis. Activation is only dependent on a 15 bp fragment residing between -209 and -195 and containing an SPH motif. In vitro, this element forms a complex with a nuclear protein which is different from the TEF-1 transcriptional activator that binds the SV40 Sph motifs. This AE is versatile since it shows capacity of activating a variety of genes in vivo, including U1 and U6 snRNAs and HSV thymidine kinase. Unexpectedly for an snRNA-related gene, the tRNA((Ser)Sec) is deprived of octamer or octamer-like motifs. The X.laevis tRNA((Ser)Sec) gene represents the first example of a Pol III snRNA-type gene whose activation of transcription is completely octamer-independent.
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PMID:Optimal tRNA((Ser)Sec) gene activity requires an upstream SPH motif. 131 Oct 68

Herpes simplex virus type 1 (HSV-1) infection induces expression of the human immunodeficiency virus type 1 (HIV-1) provirus in the chronically infected T-cell line ACH-2. The HSV-1-mediated induction correlates with the appearance of two NF-kappa B-specific proteins of 55 and 85 kDa in the nucleus and with the binding of 50-kDa nuclear protein to the LBP-1 binding site of the untranslated leader sequence of the HIV-1 long terminal repeat. The HSV-1-induced LBP-1 binding protein, designated HLP-1, is present exclusively in HSV-1-infected, but not in phorbol-12-myristate-13-acetate- or tumor necrosis factor alpha-treated ACH-2 cells. Both the NF-kappa B and LBP-1 target sequences, when inserted either alone or together 5' of a heterologous minimal promoter (thymidine kinase), confer inducibility by HSV-1 infection in a transient transfection assay. Thus, it appears that the HSV-1-mediated activation of HIV-1 provirus is brought about by the binding of both NF-kappa B and HLP-1 specific proteins to two distinct regions of HIV-1 long terminal repeat.
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PMID:Herpes simplex virus type 1-mediated induction of human immunodeficiency virus type 1 provirus correlates with binding of nuclear proteins to the NF-kappa B enhancer and leader sequence. 131 71

The proopiomelanocortin (POMC) gene is highly expressed in adult mouse pituitary anterior lobe corticotrophs and intermediate lobe melanotrophs. To identify the DNA elements important for this tissue-specific expression, we analyzed a series of POMC reporter genes in transgenic mice. A DNA fragment containing rat POMC 5'-flanking sequences from -323 to -34 recapitulated both basal pituitary cell-specific and hormonally stimulated expression in adult mice when fused to a heterologous thymidine kinase promoter. Developmental onset of the reporter gene expression lagged by 1 day but otherwise closely paralleled the normal ontogeny of murine POMC gene expression, including corticotroph activation at embryonic day 14.5 (E14.5) followed by melanotroph activation at E15.5 to E16.5. AtT20 corticotroph nuclear protein extracts interacted with three specific regions of the functional POMC promoter in DNase I protection assays. The positions of these protected sites were -107 to -160 (site 1), -182 to -218 (site 2), and -249 to -281 (site 3). Individual deletions of these footprinted sites did not alter transgene expression; however, the simultaneous deletion of sites 2 and 3 prevented transgene expression in both corticotrophs and melanotrophs. Electrophoretic mobility shift and Southwestern (DNA-protein) assays demonstrated that multiple AtT20 nuclear proteins bound to these footprinted sites. We conclude that the sequences between -323 and -34 of the rat POMC gene promoter are both necessary and sufficient for correct spatial, temporal, and hormonally regulated expression in the pituitary gland. Our data suggest that the three footprinted sites within the promoter are functionally interchangeable and act in combination with promoter elements between -114 and -34. The inability of any reporter gene construction to dissociate basal and hormonally stimulated expression suggests that these DNA elements are involved in both of these two characteristics of POMC gene expression in vivo.
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PMID:Identification of DNA elements cooperatively activating proopiomelanocortin gene expression in the pituitary glands of transgenic mice. 150 98

The epidermal growth factor (EGF) receptor (EGFR) promoter is negatively regulated by thyroid hormone and retinoic acid. This regulation can be mapped to a 36-basepair GC-rich region of the promoter (EGFR P/E) that functions autonomously as a promoter and an enhancer when placed in front of the thymidine kinase gene TATA element. Direct high affinity binding of the thyroid hormone receptor (T3R) to this element requires a nuclear protein. Through ion exchange chromatography and gel filtration of HeLa nuclear extract, this activity was identified as a protein of approximately 67 kilodaltons. This protein did not bind to DNA alone, but greatly augmented T3R binding to the EGFR P/E sequence in gel mobility shift and DNA precipitation assays. When combined with the T3R auxillary protein (TRAP), the T3R migrated as a larger complex on the DNA. Chemical cross-linking identified this complex as a heterodimer between T3R and TRAP. T3R-TRAP binds to a 7-basepair site in the EGFR P/E (GGGACTC) that has weak homology to a consensus thyroid response element half-site. Thus, on this element, T3R-TRAP heterodimers contact the DNA primarily on a single site that comprises an inhibitory thyroid response element.
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PMID:A nuclear protein is required for thyroid hormone receptor binding to an inhibitory half-site in the epidermal growth factor receptor promoter. 158 25

Epstein-Barr virus (EBV) nuclear protein 2 (EBNA-2) is essential for B-lymphocyte growth transformation. EBNA-2 upregulates mRNAs encoding CD23, a B-lymphocyte surface protein closely associated with EBV-induced growth transformation. To further investigate this EBNA-2 effect, we searched in the genomic DNA spanning the type a and type b CD23 mRNA start sites for a cis-acting fragment that would render a promoter transactivatable by EBNA-2. An 800-bp CD23 DNA fragment (-335 to +465 relative to the type a CD23 mRNA start site) conferred EBNA-2 responsiveness to the herpes simplex virus thymidine kinase (TK) promoter when transfected into EBV-negative B-lymphoma cells. Deletional analysis identified a -275/-89 subfragment that was EBNA-2 responsive when cloned in either orientation and at variable distances upstream of the heterologous promoter. EBNA-2 and the cis-acting CD23 element increased TK-promoted mRNA and did not alter the herpes simplex virus TK promoter transcription start site. As expected, a type a CD23 promoter (-335/+80) which contained the EBNA-2-responsive element was transactivated by EBNA-2. As in EBV infection and stable EBNA-2 transfection, the CD23 DNA element in cis with heterologous or homologous promoters was less responsive to type 2 than to type 1 EBNA-2, whereas the EBNA-2-responsive DNA fragment from the EBV latent membrane protein 1 promoter was more responsive to the type 2 EBNA-2. These experiments delineate a 186-bp, EBNA-2-responsive cell DNA fragment and provide firm evidence that EBNA-2 transactivates transcription of cell genes. The greater type 1 versus type 2 EBNA-2 responsiveness of the CD23 promoter and the lack of a similar effect on the latent membrane protein 1 promoter is consistent with the hypothesis that greater cell gene transactivation by type 1 EBNA-2 is the basis for the more efficient growth-transforming properties of type 1 EBV.
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PMID:Epstein-Barr virus nuclear protein 2 transactivates a cis-acting CD23 DNA element. 164 18

Expression of prostate-specific antigen (PA) mRNA was tested at various time periods after incubation of the human prostate tumor cell line LNCaP with the synthetic androgen R1881. Androgen-stimulated expression was observed within 6 h after addition of R1881 to the cells. Run-on experiments with nuclei isolated from LNCaP cells showed that expression of the PA gene could be regulated by R1881 on the level of transcription. DNase I footprints of the promoter region of the PA gene (-320 to +12) with nuclear protein extracts from LNCaP cells showed at least four protected regions. The protected areas include the TATA-box, a GC-box sequence, and a sequence AGAACAgcaAGTGCT at position -170 to -156, which closely resembles the reverse complement of the consensus sequence GGTACAnnnTGTTCT for binding of the glucocorticoid receptor and the progesterone receptor. Fragments of the PA promoter region were cloned in front of the chloramphenicol acetyltransferase (CAT) reporter gene and cotransfected with an androgen receptor expression plasmid into COS cells in a transient expression assay. CAT activity of COS cells grown in the presence of 1 nM R1881 was compared to untreated controls. A 110-fold induction of CAT activity was found if a -1600 to +12 PA promoter fragment was used in the construct. By further deletion mapping of the PA promoter a minimal region (-320 to -155) was identified as being essential for androgen-regulated gene expression. Mutation of the sequence AGAACAgcaAGTGCT (at -170 to -156) to AAAAAAgcaAGTGCT almost completely abolished androgen inducibility of the reporter gene constructs. One or more copies of the sequence AGAACAgcaAGTGCT cloned in front of a thymidine kinase promoter-CAT reporter gene confers androgen regulation to the reporter gene. These findings provide strong evidence for transcription regulation of the PA gene by androgens via the sequence AGAACAgcaAGTGCT. Interestingly, in addition to the AGAACAgcaAGTGCT element, an upstream region (-539 to -320) is needed for optimal androgen inducibility of the PA promoter.
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PMID:The promoter of the prostate-specific antigen gene contains a functional androgen responsive element. 172 87

The 5' flanking region of the mouse renin genes (Ren-1d and Ren-2d) contains two motifs that are homologous to known negative regulatory elements (NREs). Ren-2d has a 150-base-pair (bp) insertion 5' to the upstream putative NRE (NRE-1), which is lacking in Ren-1d. We tested the functionality of these sequences by using site-directed mutagenesis to delete individually each putative NRE from Ren-1d and to delete the 150-bp insertion from Ren-2d. We examined the effect of these mutations on the expression of the reporter gene chloramphenicol acetyltransferase, which was expressed from a truncated thymidine kinase promoter fused to the renin regulatory region. This plasmid was transfected into human choriocarcinoma JEG-3 cells. Only the upstream NRE (positions -619 to -597) was found to be functional in Ren-1d. The deletion of a 150-bp insertion from Ren-2d resulted in the suppression of chloramphenicol acetyltransferase activity to the level of Ren-1d expression. These data suggest that the upstream NRE that is functional in Ren-1d, but not in Ren-2d, may be partly responsible for differential expression of the renin genes in various tissues. The molecular mechanism of the NRE was examined by studying its interaction with nuclear proteins in submandibular gland and JEG-3 cells by gel-mobility-shift assays. Specific nuclear protein binding was observed only to the upstream NRE and the molecular mass of this protein was approximately 72 kDa as determined by Southwestern blot analysis. Thus our results suggest that both Ren-1d and Ren-2d conserve a cis-acting NRE in the 5' flanking region. In Ren-1d, this NRE could bind a specific nuclear protein resulting in the inhibition of Ren-1d expression in these tissues. On the other hand, the NRE in Ren-2d is nonfunctional due to interference by an adjacent 150-bp insertion.
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PMID:Identification of a negative regulatory element involved in tissue-specific expression of mouse renin genes. 173 3

The regulation by tumor necrosis factor alpha (TNF) of its own promoter has been investigated by transient transfection and nuclear protein binding assays. In human K652 erythroleukemia cells TNF produced an 8-10-fold activation of the human TNF promoter linked to the chloramphenicol acetyltransferase gene. The TNF-responsive element was localized to the -125 to -82 region by examining the TNF activation in 5'-deletion or site-directed mutants of the TNF promoter and by demonstrating that the -125 to -82 fragment confers TNF responsiveness to the thymidine kinase promoter. This region contains a palindrome, 5' TGAGCTCA 3', that resembles the consensus binding sequences for the transcription factors, activator protein-1 (AP-1), cyclic AMP-responsive element binding protein (CREB), and activation transcription factor (ATF). An internal deletion in the palindrome abolished the TNF responsiveness, whereas known AP-1 and CREB/ATF elements were unresponsive to TNF. In band shift analyses a nuclear factor from U937 cells specifically bound to the -125 to -82 TNF-responsive fragment in or near the palindromic sequence. Oligonucleotides containing AP-1 or CREB/ATF sites did not effectively compete for the binding, indicating that the U937 cell factor is different from these factors. Anti-c-fos antiserum did not affect binding of the U937 cell factor, whereas anti-c-jun antiserum did block its binding, indicating that either c-jun or a protein antigenically related to c-jun is a component of the factor. These results suggest that the TNF-responsive element is not activated by AP-1 or CREB in U937 cells and that a novel DNA binding factor is important for constitutive and inducible TNF gene expression.
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PMID:Identification of a tumor necrosis factor-responsive element in the tumor necrosis factor alpha gene. 182 93

The macrophage colony-stimulating factor (M-CSF) is required for the growth and differentiation of mononuclear phagocytes. Previous studies have demonstrated that tumor necrosis factor (TNF) induces transcription of the M-CSF gene in human myeloid cells. The present work examined the effects of TNF on cis-acting elements in the M-CSF promoter. Deleted forms of the M-CSF promoter were linked to the chloramphenicol acetyltransferase (CAT) gene and transfected by electroporation into HL-60 promyelocytic leukemia cells. The results demonstrate that an enhancer responsive to TNF stimulation is located between positions -406 and -344 upstream to the transcription start site. The fragment from positions -419 to -304 was cloned 5' to the heterologous thymidine kinase (TK) promoter and linked to the CAT gene. Both orientations of this fragment enhanced TK-promoter activity in TNF-treated HL-60 cells. The results of gel mobility shift assays with the -419 to -304 fragment demonstrate binding of a constitutive nuclear protein. A TNF-inducible protein also bound to this fragment and resulted in a different mobility pattern. Binding of the TNF-induced nuclear protein to the -419 to -304 fragment was inhibited by an oligonucleotide containing the nuclear factor-kappa B (NF-kappa B) consensus sequence. DNA footprinting demonstrated protection of an NF-kappa B binding site at positions -377 to -368. Methylation interference assays showed that the TNF-induced protein made contact points with guanine residues in the same NF-kappa B sequence. Taken together, the findings provide evidence for involvement of an NF-kappa B-like factor in transcriptional regulation of the M-CSF gene.
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PMID:Involvement of a nuclear factor-kappa B-like protein in induction of the macrophage colony-stimulating factor gene by tumor necrosis factor. 191 81

We have investigated the effect of Epstein-Barr virus nuclear antigen 1 (EBNA-1), a nuclear protein encoded by EBV, on herpes simplex virus type 1 (HSV-1) infection either in cells constitutively expressing EBNA-1 or in transient expression assays. Rat-1 cells and rat embryo fibroblasts (REF) immortalized by c-myc or E1A were transfected with a specific EBV DNA fragment coding for EBNA-1. Cloned cell lines which constitutively expressed this antigen were infected with HSV-1. Our results indicate that in EBNA-1-expressing cells, virus growth was higher than in control cells for different virus strains or rodent cell lines. This increase was maximal when cells were infected at low multiplicity, as determined by virus growth, and correlated with the stimulation of viral DNA synthesis. REF + c-myc and Vero cells were cotransfected by an EBNA-1 expression vector driven by Moloney murine leukaemia virus LTR and HSV-1 immediate-early (alpha 0) or early thymidine kinase upstream promoter regulatory regions linked to chloramphenicol acetyltransferase (CAT) coding sequences as effectors. In both cell lines, stimulation of CAT expression by EBNA-1 was observed only with the immediate-early promoter. These results suggest that EBNA-1 can transactivate immediate-early HSV-1 expression.
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PMID:Herpes simplex type 1 activation by Epstein-Barr virus nuclear antigen 1. 215 38


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