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)

DNase I footprint analysis, using total HeLa cell nuclear extract and purified transcription factor Sp1, was carried out to determine the various protein binding sites within the human thymidine kinase promoter. The promoter has two separate CCAAT elements and multiple Sp1-binding sites, as well as at least one undefined protein binding site. Detailed analysis of protein binding to the two CCAAT elements showed that changing the spacing between the two CCAAT elements altered both protein binding to the distal CCAAT element as well as promoter activity. Both CCAAT elements can act as functional transcription elements, but are not oriented for optimal promoter strength in the human tk promoter. Our studies show that a promoter fragment that has been previously shown to be the minimal region to maintain a serum responsive promoter regulation apparently contains only a single Sp1-binding domain and the more distal of the CCAAT elements.
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PMID:Protein binding sites within the human thymidine kinase promoter. 154 2

The herpes simplex virus type 1 (HSV-1) ICP4 protein is a transcriptional activator of many eucaryotic RNA polymerase II promoters. The HSV-1 thymidine kinase gene (tk) promoter is induced by ICP4 and contains binding sites for the cellular transcription factors TFIID, Sp1, and CCAAT-binding proteins, each of which affects expression of the tk gene. In this study, the effects of mutations in these sites on the transcription of tk in the presence and absence of ICP4 were determined during viral infection. Only the TATA box was necessary for efficient expression in the presence of ICP4; however, ICP4 apparently can still induce tk transcription even when the TATA box is disrupted. Alteration of the Sp1 sites had a minor effect on ICP4-induced expression in comparison to a large effect in the absence of ICP4, indicating that ICP4 can operationally substitute for the function of the transcription factor Sp1. In addition, tk was still expressed with the kinetics of an early gene in the absence of binding sites for Sp1 and CCAAT-binding proteins.
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PMID:Herpes simplex virus transactivator ICP4 operationally substitutes for the cellular transcription factor Sp1 for efficient expression of the viral thymidine kinase gene. 184 84

The mouse gene Krox-24 is transiently activated during cell cycle reentry. It encodes a protein with three zinc fingers similar to those of the transcription factor Sp1. Here we present a biochemical characterization of the gene products. Krox-24 mRNA is translated into two proteins of 82 and 88 kilodaltons, designated p82Krox-24 and p88Krox-24, respectively. p82Krox-24 is initiated at the first AUG codon of the open reading frame, whereas synthesis of p88Krox-24 starts at a non-AUG codon located upstream. Both proteins were synthesized in HeLa cells infected with recombinant vaccinia viruses expressing Krox-24 cDNAs. Under these conditions, they were found phosphorylated on serine residues and glycosylated. The availability of the proteins made possible the determination of the DNA recognition sequence. In vitro, Krox-24 bound specifically to the sequence 5'-GCG(C/G)GGGCG-3'. This sequence is similar but not identical to the Sp1 target sequence. Insertion of an oligomer for the binding site in cis, close to the herpes simplex virus thymidine kinase promoter, rendered this promoter responsive to Krox-24. Krox-24 is therefore a sequence-specific transcriptional activator. Krox-24-binding sites were found upstream of several serum-inducible genes, raising the possibility that Krox-24 is involved in the regulation of these genes.
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PMID:The serum-inducible mouse gene Krox-24 encodes a sequence-specific transcriptional activator. 211 74

A 440-base-pair fragment of African green monkey genomic DNA shares homology with the transcriptional regulatory region of simian virus 40 (SV40) and has been reported to direct transcription in vivo. We find that two regions within this fragment bind the promoter-specific cellular transcription factor Sp1 and are protected in DNase protection ("footprinting") experiments. As in SV40, binding occurs in regions containing multiple copies of the sequence GGGCGG. These regions, when fused to the proximal, or "TATA box," element of the herpes simplex virus thymidine kinase promoter, are able to direct Sp1-dependent transcription in vitro. The finding that Sp1 is capable of productive interaction with sequences taken from a cellular promoter supports the idea that Sp1 may play a role in modulating transcription of cellular genes.
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PMID:Transcription factor Sp1 recognizes promoter sequences from the monkey genome that are simian virus 40 promoter. 299 98

We have previously shown that the Tat protein of the human immunodeficiency virus type 1 (HIV-1) is a modular transcriptional activator that can be targeted upstream of either a synthetic promoter or the intact HIV promoter to activate transcription. This activation was shown to be largely dependent on the presence of consensus binding sites for the cellular transcription factor Sp1. Since the use of heterologous promoters may provide further insight into Tat-mediated transactivation, we have analyzed the transactivation of the thymidine kinase promoter of herpes simplex virus by Tat and by the acidic transcriptional transactivator VP16. The effects of mutations of defined upstream promoter elements show that Tat transactivation is dependent on Sp1 binding sites in a site-specific manner. In contrast, transactivation by the acidic transactivator VP16 is completely independent of any of the defined promoter elements upstream of the TATA box. These results suggest that Tat and the classically defined modular acidic transcriptional activators have different modes of transactivation. In addition, the substitution of the HIV-1 TATA box for the thymidine kinase TATA box substantially increases Tat transactivation, indicating that Tat transactivation may also ultimately involve TATA box-associated cellular transcription factors.
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PMID:Activation of a heterologous promoter by human immunodeficiency virus type 1 Tat requires Sp1 and is distinct from the mode of activation by acidic transcriptional activators. 841 86

Genomic clones containing the gene for the glutathione peroxidase-like androgen-regulated murine epididymal protein of 24 kilodaltons (arMEP24) were isolated. A 9-kilobase DNA fragment was sequenced and found to contain the entire coding region of the gene, which is divided into five exons. The exact sizes and boundaries of the exon blocks were deduced by comparison with the cDNA sequence. One major and four weak transcription initiation sites in the epididymis were localized by primer extension. The promoter of the gene does not contain a conventional TATA box immediately up-stream of the start site; rather, the sequence TATCA occurs at residue -35. Two CAAT boxes in opposite orientation and two putative binding sites for the transcription factor Sp1 were identified up-stream of the TATA-like box. To localize the cis-acting sequences responsible for androgen regulation of expression, fragments of the arMEP24 gene promoter region were cloned in front of the luciferase (LUC) reporter gene and cotransfected with an androgen receptor expression vector into CV-1 cells in a transient assay. LUC activities of CV-1 cells grown in the presence of various concentrations of 5 alpha-dihydrotestosterone were compared to LUC activities of untreated controls. The DNA fragment containing up to 200 nucleotides up-stream from the major transcription start site was sufficient for the full promoter activity, but not for the responsiveness to androgen induction. Depending on the 5 alpha-dihydrotestosterone concentration, a 2- to 4-fold induction of LUC activity was found if a -1797 to -167 arMEP24 gene fragment was used linked to the reporter gene driven by either the homologous promoter or the heterologous thymidine kinase promoter. Two or three copies of the imperfect palindromic sequence TGTTGAgagAGAACA, found at position -896 to -882 in the gene and resembling the consensus steroid hormone-responsive element, are able to confer androgen regulation to the thymidine kinase promoter independently of their orientation. These findings support evidence that transcriptional regulation of the arMEP24 gene occurs via the sequence TGTTGAgagAGAACA. Homologies found in the sequence up-stream of the promoter with several putative binding sites for erythroid-specific trans-acting regulatory proteins are discussed. Finally, the arMEP24 gene is located by in situ hybridization in the [A2-A4] region of mouse chromosome 13.
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PMID:Structural organization and regulation of the gene for the androgen-dependent glutathione peroxidase-like protein specific to the mouse epididymis. 846 39

Within the region around 150 bp upstream of the initiation codon, which was previously shown to suffice for growth-regulated expression, the murine thymidine kinase gene carries a single binding site for transcription factor Sp1; about 10 bp downstream of this site, there is a binding motif for transcription factor E2F. The latter protein appears to be responsible for growth regulation of the promoter. Mutational inactivation of either the Sp1 or the E2F site almost completely abolishes promoter activity, suggesting that the two transcription factors interact directly in delivering an activation signal to the basic transcription machinery. This was verified by demonstrating with the use of glutathione S-transferase fusion proteins that E2F and Sp1 bind to each other in vitro. For this interaction, the C-terminal part of Sp1 and the N terminus of E2F1, a domain also present in E2F2 and E2F3 but absent in E2F4 and E2F5, were essential. Accordingly, E2F1 to E2F3 but not E2F4 and E2F5 were found to bind sp1 in vitro. Coimmunoprecipitation experiments showed that complexes exist in vivo, and it was estabilished that the distance between the binding sites for the two transcription factors was critical for optimal promoter activity. Finally, in vivo footprinting experiments indicated that both the sp1 and E2F binding sites are occupied throughout the cell cycle. Mutation of either binding motif abolished binding of both transcription factors in vivo, which may indicate cooperative binding of the two proteins to chromatin-organized DNA. Our data are in line with the hypothesis that E2F functions as a growth- and cell cycle regulated tethering factor between Sp1 and the basic transcription machinery.
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PMID:Interaction of Sp1 with the growth- and cell cycle-regulated transcription factor E2F. 865 41

The transcription factor Sp1 plays a key role in the activation of many cellular and viral gene promoters, including those that are regulated during the cell cycle. However, recent evidence indicates that Sp1 belongs to a larger family of factors which bind G/C box elements in order to either activate or repress transcription. Sp3, a member of this family, functions to repress transcriptional activation in two viral promoters, most likely by competing with Sp1 for GC box/Sp binding sites. However, the physiological role of Sp3 in the repression of endogenous cellular promoters has not been experimentally addressed. In the present study, we analyze the activity and binding of Sp3 on several eukaryotic promoters that contain G/C boxes and are known to be regulated during cellular proliferation and the cell cycle. Using antibodies specific for Sp1 and Sp3, we observe that both of these factors localize to the cell nucleus and have a similar, dispersed subnuclear distribution. Further, using gel mobility shift assays, we show that both Sp1 and Sp3 interact specifically with the histone H4 promoter. Transient cotransfections of Drosophila cells with Sp1 and Sp3 expression vectors and with the histone H4, thymidine kinase (TK), or dihydrofolate reductase (DHFR) promoters show that only the DHFR promoter, containing multiple functional GC boxes, displays Sp3 repression of Sp1 activation. In contrast, the single G/C boxes within the histone H4 or TK promoters, which confer transcriptional activation via Sp1 binding, are not responsive to repression by Sp3. Therefore, we demonstrate that the endogenous cellular DHFR promoter is selectively responsive to Sp3 repression. The data suggest that Sp3 may contribute to the control of proliferation- and/or cell-regulated promoters depending upon the context and/or number of functional Sp1 binding sites.
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PMID:Sp1 trans-activation of cell cycle regulated promoters is selectively repressed by Sp3. 884 79