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)

At early times after infection with herpes simplex virus, transcription from beta-promoters is initiated only in the presence of a functional 174,000 Mr phosphoprotein (ICP4), encoded by an immediate early (alpha) gene (IE4). A transient expression assay was used to analyze the requirement for two (ICP4 and ICP0) of the five alpha-gene products in the transcriptional regulation of model alpha and beta-gene promoters. These studies reveal that cells cotransfected with plasmids containing the alpha-gene sequences for infected cell proteins (ICPs) 4 and 0 and a thymidine kinase (TK, a beta-gene) gene or the thymidine kinase promoter fused to a chloramphenicol acetyltransferase (CAT) cassette accumulate 10 to 20-fold more RNA or exhibit 10 to 20-fold more CAT activity than cells cotransfected with a plasmid encoding either alpha-gene protein and a thymidine kinase indicator gene. Functional ICP4 is required for enhanced transcriptional activation in the transient expression assay system. It is also required for the uniform dispersal of ICP0 throughout the nucleus as shown by immunofluorescence staining analysis of transfected cells. Two alpha-promoter-CAT fusions were used as targets to study what effects ICP4, ICP0 and Vmw65 (the virion-associated alpha-gene transactivator) have on expression from alpha-promoters that contain all of the sequences that confer alpha-gene regulation, or only the core sequence governing basal level expression. We conclude that ICP4 can activate alpha-gene expression from the core sequence and, depending on its abundance, activate or repress expression from a promoter containing the sequences required for alpha-gene regulation. Independent of these alpha-regulatory sequences cotransfection with low levels of sequences encoding both ICP0 and ICP4 activate expression. At higher ratios of effector (both ICP4 and ICP0) the target accumulation of CAT activity decreases. Although a ts allele of IE4 (cloned from the mutant virus tsK) does not activate alpha-gene expression it can enhance the ability of ICP0 to activate a target containing alpha-regulatory sequences. Virus studies involving tsK support the conclusion that functional ICP4 is required to activate beta-promoters and to repress expression from alpha-promoters and help to explain the pleiotropic effects of the tsK mutation. These analyses have also revealed the presence of a novel RNA species that overlaps the sequences encoding ICP0. Our results suggest that co-ordinate regulation of HSV gene expression is mediated by the functional interaction of at least two alpha-gene products, ICP0 and ICP4.
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PMID:Co-ordinate regulation of herpes simplex virus gene expression is mediated by the functional interaction of two immediate early gene products. 302 83

A 94-kilodalton phosphoprotein known as IE94 is the only viral polypeptide synthesized in abundance under immediate-early conditions after infection by cytomegalovirus (CMV) strain Colburn in either permissive primate or nonpermissive rodent cells. The IE94 gene, which maps at coordinates 0.71 to 0.73 in the viral genome, contains a large intron in the 5' leader sequence, and its promoter regulatory region contains novel, multiple-palindromic, repeated elements. Two recombinant plasmids (pTJ148 and pTJ198) containing the 10.5-kilobase-pair HindIII-H DNA fragment from CMV (Colburn) were transfected into mouse Ltk- cells, by either linked or unlinked coselection in hypoxanthine-aminopterin-thymidine medium, together with herpes simplex virus thymidine kinase genes. With both procedures, constitutive synthesis of the IE94 immediate-early protein was detected in pools of Ltk+ cells by immunoprecipitation. Subsequently, we isolated a clonal Ltk+ cell line which expressed the [35S]methionine-labeled IE94 polypeptide in sufficient abundance to be visualized directly in autoradiographs after gel electrophoresis of total-cell-culture protein extracts. The IE94 polypeptide synthesized in the transfected cells was indistinguishable in size and overall net charge from that produced in virus-infected cells. In addition, the IE94 protein expressed in LH2p198-3 cells was phosphorylated (presumably by a cellular protein kinase) and generated similar phosphopeptide patterns after partial tryptic digestion to those obtained with the CMV IE94 protein from infected cells. The cell line contained two to four stably integrated copies of the IE94 gene and synthesized a single virus-specific mRNA of 2.5 kilobases detectable on Northern blots. A new antigen, detectable by indirect anticomplement immunofluorescence with monoclonal antibody against the human CMV IE68 protein, was present in the nuclei of more than 95% of the LH2p198-3 cells. This evidence suggests that (unlike most herpesvirus genes) the CMV IE94 gene, together with its complex promoter and spliced mRNA structure, may contain all of the regulatory elements necessary for strong constitutive expression in mammalian cells in the absence of other viral factors.
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PMID:Abundant constitutive expression of the immediate-early 94K protein from cytomegalovirus (Colburn) in a DNA-transfected mouse cell line. 609 48

The mouse lactoferrin gene responded to forskolin, 12-O-tetradecanoyl phorbol-13-acetate, and epidermal growth factor (EGF) stimulation via two adjacent enhancer elements, the cAMP response element (CRE) and EGF response element (EGFRE), collectively referred to as the mitogen response unit. In this report, we examined the minimal promoter and enhancer elements of the mouse lactoferrin gene that are required for EGF-induced transcriptional activation. We found that the CRE and noncanonical TATA box (ATAAA) are the minimal promoter elements for basal activity of the chloramphenicol acetyltransferase (CAT) reporter construct whereas the EGFRE is needed for an additional activity induced by EGF in transiently transfected human endometrial carcinoma RL95-2 cells (RL95-2). The EGFRE, however, did not function in heterologous promoters [SV 40 and thymidine kinase (TK)]. Therefore, EGF-stimulated lactoferrin gene activity is promoter specific in RL95-2 cells. In transiently transfected cells, EGF and forskolin showed synergistic effects on the CAT reporter that contained both response elements. Mutation made at either element or insertion of extra nucleotides between the two elements severely affected EGF-stimulated activity. Nuclear protein prepared from RL95-2 cells formed three complexes (A, B, and C) with the oligonucleotides containing both EGFRE and CRE in electrophoretic mobility shift assay. A new complex (E) was detected with the nuclear protein of EGF-treated cells. By oligonucleotide competition experiments, we demonstrated that the complex E was generated by protein bound to CRE. EGF-induced binding activity could be abolished by calf intestinal alkaline phosphatase but not by the protein synthesis inhibitor, cycloheximide. Therefore, binding of a preexisting phosphoprotein to the CRE region could be one of the requirements for EGF-induced mouse lactoferrin gene promoter activity.
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PMID:Promoter-specific activation of mouse lactoferrin gene by epidermal growth factor involves two adjacent regulatory elements. 877 33

The equine herpesvirus 1 (EHV-1) homolog of the herpes simplex virus type 1 (HSV-1) tegument phosphoprotein, alphaTIF (Vmw65; VP16), was identified previously as the product of open reading frame 12 (ORF12), was shown to trans-activate immediate-early (IE) gene promoters, and was described as a 60-kDa virion component designated ETIF. However, the ETIF promoter region and transcription initiation site were not identified. The poly(A) signal of the gene 11 (UL49 homolog) lies just upstream of the first ETIF translation initiation codon, indicating that the first ATG may not be used for initiating ETIF translation. Another in-frame translation initiation codon (ATG2) is located 88 bp downstream of the first ETIF initiation codon (ATG1). Western blot analysis showed that the expressed ETIF protein migrated in SDS-PAGE with an apparent molecular mass of approximately 56 kDa, the same molecular weight identified in SDS-PAGE analysis of the KyD EHV-1 virion preparations. The ETIF expression vector pCETIF, which contains ATG2, trans-activated the IE promoter more efficiently than the pC12 containing both ATG1 and ATG2. S1 nuclease analyses mapped the 5' initiation site of the 1.4-kb transcript approximately 17 to 21 nt downstream of the ATG1. The nucleotide sequence upstream of the ATG1 did not have any promoter activity, while the nucleotide sequence upstream of the ATG2 had promoter activity. In transient transfection assays, the pETIFM2 vector, which was mutated in the ATG2, did not trans-activate the IE promoter; however, the pETIFM1 vector, which was mutated in the ATG1, trans-activated the IE promoter. These results demonstrated that the ATG2 of the ETIF ORF is the ETIF translation initiation codon. ETIF trans-activated only the IE promoter, not early (EICP0, EICP22, EICP27, and thymidine kinase) or late (IR5) promoters, confirming that EICP0, EICP22, and EICP27 are early genes.
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PMID:Molecular characterizations of the equine herpesvirus 1 ETIF promoter region and translation initiation site. 1144 76

The sole immediate-early (IE) gene of equine herpesvirus 1 encodes a 1,487-amino-acid (aa) regulatory phosphoprotein that independently activates expression of early viral genes. Coimmunoprecipitation assays demonstrated that the IE protein physically interacts with the general transcription factor TFIIB. Using a variety of protein-binding assays that employed a panel of IE truncation and deletion mutants expressed as in vitro-synthesized or glutathione S-transferase fusion proteins, we mapped a TFIIB-binding domain to aa 407 to 757 of the IE protein. IE mutants carrying internal deletions of aa 426 to 578 and 621 to 757 were partially defective for TFIIB binding, indicating that aa 407 to 757 may harbor more than one TFIIB-binding domain. The interaction between the IE protein and TFIIB is of physiological importance, as evidenced by transient-cotransfection assays. Partial deletion of the TFIIB-binding domain within the IE protein inhibited its ability to activate expression of the viral thymidine kinase gene, a representative early promoter, and of the IR5 gene, a representative late promoter, by greater than 20 and 50%, respectively. These results indicate that the interaction of the IE protein with TFIIB is necessary for its full transactivation function and that the IE-TFIIB interaction may be part of the mechanism by which the IE protein activates transcription.
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PMID:Mapping the sequences that mediate interaction of the equine herpesvirus 1 immediate-early protein and human TFIIB. 1158 90

E2F transcription factors play an essential role in cell proliferation and apoptosis and their activity is frequently deregulated in human cancers. In a yeast two-hybrid screen we identified a novel E2F-binding protein. Due to its strong phosphorylation we named it EAPP (e2F-associated phosphoprotein). EAPP is localized in the nucleus and interacts with E2F-1, E2F-2, and E2F-3, but not with E2F-4. Examination of a number of human cell lines revealed that EAPP levels are elevated in most transformed cells. Moreover, EAPP mRNA was detected in all investigated human tissues in varying amounts. EAPP is present throughout the cell cycle but disappears during mitosis. In transfection assays with reporters controlled by either an artificial E2F-dependent promoter or the murine thymidine kinase promoter, EAPP increased the activation caused by E2F-1 but not by E2F-4. Surprisingly, the promoter of the p14(ARF) gene, which was also activated by E2F-1, became repressed by EAPP. Overexpression of EAPP in U2OS cells resulted in a significant increase of cells in S-phase, whereas RNAi-mediated knock down of EAPP reduced the fraction of cells in S-phase. Taken together, these data suggest that EAPP modulates E2F-regulated transcription, stimulates proliferation, and may be involved in the malignant transformation of cells.
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PMID:EAPP, a novel E2F binding protein that modulates E2F-dependent transcription. 1571 52