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 sea urchin early H2A histone gene, like the other four members of the repeating units, is transiently expressed during very early development. To investigate the mechanisms underlying the faithful expression of the early H2A gene, we focused our attention on the modulator element. We showed by DNase I cleavage protection patterns that the modulator includes the upstream sequence element 1 (USE1) and mapped at nucleotides -137 to -108 in the early H2A gene promoter. Functional tests conducted by microinjection into sea urchin embryos then showed that the modulator element binds the transcriptional factor called modulator-binding factor 1 (MBF-1). We found in fact that coinjection of an excess of the MBF-1-binding site, either as the modulator or as the USE1, efficiently impaired the activity of the H2A promoter. An unexpected finding was the expression of the reporter gene from the early H2A promoter at the gastrula stage of embryonic development, when the early histone genes are transcriptionally silent. In addition, we also found that the modulator element was active at the gastrula stage. The potential enhancer activity of the modulator was tested by microinjecting several constructs containing single or multiple copies of the modulator element placed 5' or 3' to a thymidine kinase gene (tk) promoter in both sea urchin embryos and Xenopus laevis oocytes and determining the expression of a reporter chloramphenicol acetyltransferase gene under the control of the linked tk promoter. We found that an oligonucleotide bearing the MBF-1-binding site activates the expression of the reporter gene independently of the position and orientation. We conclude that the modulator binds the MBF-1 activator and that it is a transcriptional enhancer of the early H2A histone gene.
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PMID:Modulator factor-binding sequence of the sea urchin early histone H2A promoter acts as an enhancer element. 799 25

Glucocorticoids inhibit transcription of the proto-oncogene c-myc in lymphoid cells of thymic origin. To determine if this effect is associated with changes in the properties of the transcription factor E2F, extracts were prepared from control and glucocorticoid-treated P1798 murine T lymphoma cells, and the macromolecular state of E2F was assessed by gel-mobility shift. Control extracts exhibit two predominant gel-mobility shift entities of which one corresponds to "free" E2F. A second entity, complex C, has properties similar to those described for the complex containing E2F, p107, cyclin A, and Cdk2. Complex C disappears after addition of dexamethasone and is replaced by complex D. The mobility of this complex and its sensitivity to SV40 T antigen suggest that complex D corresponds to an E2F-p105Rb-1 complex. Extracts from control and glucocorticoid-treated cells yield identical DNase I protection patterns on the c-myc P2 promoter. Furthermore, such extracts transcribe the c-myc P2 promoter in vitro with equal activity. The relative abundance of the E2F complexes was measured after addition of dexamethasone. Complex C disappears as cells withdraw from S phase, and complex D appears at this time. The genes encoding thymidine kinase (Tk-1) and p34cdc2 (cdc2) are regulated with kinetics similar to those observed for changes in the macromolecular state of E2F. However, regulation of c-myc expression occurs long before any change in E2F. The macromolecular state of E2F may regulate expression of genes at the G1/S boundary. However, the data are not consistent with the hypothesis that association of E2F with tumor suppressor gene products such as p107 or p105Rb-1 is relevant to glucocorticoid regulation of c-myc transcription.
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PMID:The macromolecular state of the transcription factor E2F and glucocorticoid regulation of c-myc transcription. 800 8

The cytochrome P450 gene CYP2C11, expressed in the liver of male rats, is transcriptionally regulated in a dual fashion by the sexually dimorphic secretion pattern of growth hormone. To enable analysis of transcriptional regulatory DNA elements, rat genomic sequences were cloned. DNase I hypersensitivity analysis of rat liver nuclei revealed the existence of two hypersensitive sites whose presence in the vicinity of the transcription start site correlates to high transcriptional activity of the gene. Deletion mutants of the 5' flank were fused to reporter genes and transiently transfected into HepG2 cells or into primary adult rat hypatocytes. Transfection experiments in combination with DNase I footprinting analysis in vitro led to the identification of two negative regulatory regions spanning nucleotides -1,230 to -1,188 and -409 to -368 and designated (SIL1200) and (SIL400), respectively. When placed in front of the heterologous thymidine kinase promoter, SIL1200 and SIL400 reduced the activity of the chloramphenicol acetyl transferase reporter gene to 13% and 23% of the control value, respectively. No sex-dependent binding of liver nuclear extracts to the two silencers could be detected by in vitro footprinting or gel retardation assays. However, a sex-dependent footprint consistently stronger with male liver nuclear extracts than with female extracts was observed in the -320 to -294 region. A significant level of identity was found between the DNA sequence corresponding to this footprint and that of orphan steroid receptor elements as well as with that of a basal transcription element common to several CYP2C genes. However, the identity of a potential trans-acting factor binding between -320 and -294 or response of this element to growth hormone is as yet unknown. A sex- and GH secretory profile-dependent protein-DNA interaction in vitro was observed in the -107 to -95 region. In spite of the sequence similarity that exists between this region and the consensus binding site for HNF-1, this region does not bind HNF-1 alpha. This element acted as a repressor on the heterologous thymidine kinase promoter. To date, the two silencer elements and possibly also the HNF-1-like element are the only functional elements defined in the CYP2C11 gene, and it is conceivable that induction of the gene involves derepression of the silencer elements.
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PMID:Characterization of the proximal promoter and two silencer elements in the CYP2C11 gene expressed in rat liver. 806 5

Multidrug resistance in mammalian cells is often associated with the overproduction of a membrane glycoprotein, P-glycoprotein, that is encoded by mdr genes. Multidrug resistance cell lines selected with either vinblastine, colchicine, or taxol from the drug-sensitive murine macrophage-like cell line J774.2 overexpress the mdr1a and/or mdr1b genes, and overproduce P-glycoprotein. To elucidate the mechanisms of mdr1b gene expression, the mdr1b 5'-flanking sequences have been isolated from a normal mouse liver genomic library and analyzed by gel shift and DNase I footprinting assays. These analyses have demonstrated three nuclear protein binding sites, from -82 to -59 (site 1), from -123 to -101 (site 2), and from -272 to -249 (site 3), which interact with proteins present in nuclear extracts from both sensitive and resistant cells. Although site 1 contains a partially conserved AP-2 consensus sequence, our results indicate that the nuclear protein binding to site 1 is not AP-2 protein. The sequence of site 2 is conserved in the murine mdr1a, human mdr1, and hamster pgp1 promoters. Such conservation suggests that this sequence may have an important role in mdr gene expression. The use of a transient chloramphenicol acetyltransferase expression vector containing the basal promoter for herpes simplex virus thymidine kinase (tkCAT) and either site 1 or site 2 or both revealed that the sequences of sites 1 and 2 enhanced tkCAT activity. DNase I footprinting analyses demonstrated that site 3 is recognized by human AP-1 protein, indicating that the nuclear protein binding to this site is an AP-1-like protein. These observations suggest that mdr1b gene expression is mediated by preexisting transcription factors present in sensitive and resistant cells.
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PMID:Three distinct nuclear protein binding sites in the promoter of the murine multidrug resistance mdr1b gene. 809 13

Enhancer activities have been observed in DNA fragments up to 1.36 kb long located on the 3'-side of the cluster of the three alpha-type globin-encoding genes in duck [Kretsovali et al., C.R. Acad. Sci. Paris 307 (1988) 563-568] and chicken [Knezetic and Felsenfeld, Mol. Cell. Biol. 9 (1989) 893-901]. We report here the identification of a chicken silencer element placed upstream from the three GATA-1 sites which constitute the core enhancer element in both species. This silencer element can autonomously reduce the activity of promoters for thymidine kinase and alpha D globin. Band shifts and DNase I footprinting experiments using nuclear extracts from thermosensitive avian erythroblastosis virus-transformed chicken erythroblasts led to the delineation of three sites for DNA-binding proteins within the silencer element.
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PMID:Silencer and enhancer elements located at the 3'-side of the chicken and duck alpha-globin-encoding gene domains. 810 Jul 90

Previous studies indicate that a human chorionic somatomammotropin (hCS) gene enhancer (CSEn) associated with the growth hormone (hGH) gene locus is involved in directing cell-specific expression of the hCS genes in placenta. In the current studies, we report a detailed structural analysis of this enhancer. CSEn stimulated transcription of a variety of promoters, including the hCS, human growth hormone, thymidine kinase, and Rous sarcoma virus promoters, in human choriocarcinoma cell lines (BeWo and JEG-3) but not HeLa cells or rat somatolactotrophes (GC). Maximal enhancer activity was confined to a 242-base pair DNA segment. Of several CSEn subfragments, only the En 57/242 subfragment retained activity (33.5% wild-type). The CSEn DNA sequence contained direct and inverted repeat motifs and sequences related to the SV40 enhansons, GT-IIC, GT-I, and SphI/SphII. DNase I footprint analysis revealed that most of these sites were protected by nuclear proteins derived from BeWo, JEG-3, HeLa, and GC cells. Site-specific block mutation of the GT-IIC-related and inverted repeat motifs virtually abolished enhancer activity, and mutation of all but the GT-I-related motif resulted in significant loss (30-60%) of activity. These data demonstrate that the CS enhancer is comprised of multiple elements related to SV40 enhansons that interact cooperatively to generate enhancer function.
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PMID:The human chorionic somatomammotropin gene enhancer is composed of multiple DNA elements that are homologous to several SV40 enhansons. 814 21

Angiotensinogen is shown to be produced by the liver and the hepatoma cell line HepG2. As a first step for understanding the molecular relationship between the transcriptional regulation of the angiotensinogen gene and the pathogenesis of hypertension, we have analyzed the basal promoter of the angiotensinogen gene. Chloramphenicol acetyltransferase (CAT) assays with 5'-deleted constructs showed that the proximal promoter region from -96 to +22 of the transcriptional start site was enough to express HepG2-specific CAT activity. Electrophoretic mobility shift assay and DNase I footprinting demonstrated that the liver- and HepG2-specific nuclear factor (angiotensinogen gene-activating factor [AGF2]) and ubiquitous nuclear factor (AGF3) bound to the proximal promoter element from -96 to -52 (angiotensinogen gene-activating element [AGE2]) and to the core promoter element from -6 to +22 (AGE3), respectively. The site-directed disruption of either AGE2 or AGE3 decreased CAT expression, and the sequential titration of AGF3 binding by in vivo competition remarkably suppressed HepG2-specific CAT activity. Finally, the heterologous thymidine kinase promoter assay showed that AGE2 and AGE3 synergistically conferred HepG2-specific CAT expression. These results suggest that the synergistic interplay between AGF2 and AGF3 is important for the angiotensinogen promoter activation.
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PMID:Molecular mechanism of transcriptional activation of angiotensinogen gene by proximal promoter. 816 41

By performing DNase I footprint analysis, we had identified three distinct protein binding sequences (MT1, MT2, and MT3) located on the mouse thymidine kinase (TK) upstream promoter (Dou, Q.-P., Fridovich-Keil, J. L., and Pardee, A.B. (1991) Proc. Natl. Acad. Sci. U.S.A. 88, 1157-1161). Here we report that MT2 includes an E2F-like binding site (GTTCGCGGGCAAA), as shown by the following evidence. (i) MT2 bound specifically to an affinity-purified fusion human E2F protein. (ii) Both MT2 and an authentic E2F site (TTTCGCGCGCTTT) bound specifically to similar or identical nuclear protein complexes. (iii) Formation of both these DNA-protein complexes were cell cycle-dependent: a G0/G1 phase-specific complex (E2F.G0/G1) was replaced by an S phase-specific complex(es) (E2F.S), whereas "free" E2F increased after the G1/S transition. (iv) Pulse inhibition of protein synthesis with cycloheximide interchanged these complexes with similar kinetics. (v) When MT2-shifted E2F.G0/G1, E2F.S, and free E2F were eluted and analyzed by Western blot assay using a specific antiserum to human E2F-1, two forms of murine E2F (62 and 66 kDa) were observed from all three complexes. The compositions of these MT2-bound complexes were also investigated. Studies using specific antibodies revealed that p107, a retinoblastoma-like protein, was present in both E2F-G0/G1 and E2F.S, whereas cyclin E.cyclin A.cdk2 were only present in E2F.S complex(es). These data suggest that removal of the p107-containing E2F.G0/G1 complex, a candidate repressor, from the MT2 site in late G1 may be essential for S phase-dependent transcription of the mouse TK gene.
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PMID:G1/S-regulated E2F-containing protein complexes bind to the mouse thymidine kinase gene promoter. 828 95

We have used the sequence of the cell-cycle regulatory region of human thymidine kinase promoter to study the DNA-protein interaction in human tumor and normal cells. By performing band-shift assays and DNase I footprint analysis, we have demonstrated that human tumor cells exhibited an elevated level of binding activity to the distal CCAAT box of human thymidine kinase promoter. The expression of human thymidine kinase CCAAT-binding activity was serum or independent in human tumor cells but serum dependent in normal human diploid fibroblasts. Our results present the fact that the constitutive interaction of CCAAT-binding factor with the promoter of a cell growth-controlled gene, such as thymidine kinase, is consistent with the loss of stringent cell growth regulation associated with tumorigenic phenotype.
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PMID:Constitutive overexpression of DNA binding activity to the distal CCAAT box of human thymidine kinase promoter in human tumor cell lines. 832 36

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


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