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)

Von Willebrand factor (vWF), a protein necessary for platelet adhesion and thrombus formation, is specifically synthesized in endothelial cells and in platelet precursors (megakaryocytes). We previously demonstrated that the sequences localized either in the 5'-flanking region or in the first exon of human (hu) vWF gene (vWF), which regulate the cell-specific transcription, are not conserved in the bovine counterpart. In order to look for cis-acting elements involved in the endothelial expression of bovine (bo) vWF, fragments including 113 base pairs (bp) of a sequence 5'-flanking the transcription start point (tsp, +1) and various deletions of the first 233 bp exon were linked in plasmids to the bacterial chloramphenicol (Cm) acetyltransferase gene (cat). These constructs were analyzed by transient transfection in calf pulmonary artery endothelial (CPAE), human epithelial (HeLa) from cervix and green monkey fibroblasts from kidney (COS) cells. The longest fragment, containing 229 bp of the first exon, was the most active, with identical cat expression in the three cell types. The CAT activity was equivalent to that measured by transfection of the same cells with the basal promoter (from bp -89 to +19) of hu vWF. Addition of upstream bo vWF sequences from bp -113 to -1362 resulted in progressive reduction of the activity of the -113/+229 fragment. The upstream negative regulatory domains between -1362 and -278 also repressed the heterologous thymidine kinase (tk) promoter in CPAE and HeLa cells. Comparison of results with those previously obtained by transfection of hu vWF promoter in bovine endothelial cells demonstrates that the cis-acting elements do not behave identically in bo vWF promoter. In particular, positive tissue-specific elements able to overcome the negative regulation in endothelial cells could not be found in bo vWF between bp -1362 and +229.
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PMID:Functional characterization of bovine von Willebrand factor gene promoter in bovine endothelial cells demonstrates species-specific properties. 937 Feb 73

Several lines of evidence indicate that the number of GnRH receptors (GnRH-R) and therefore, gonadotrope responsiveness to GnRH, is highly dependent upon the level of GnRH-R mRNA. To explore this aspect of regulation, we have isolated a 3.3 kb fragment encompassing the promoter region of the rat GnRH-R gene. Primer extension and RNase protection assays allowed the identification of five major transcriptional start sites located within the 110 bp region upstream of the translation start codon. Transfection experiments using the CAT reporter gene demonstrated that the 1261 bp 5' flanking region is required to direct high efficient expression in the gonadotrope-derived alphaT3-1 cell line thus contrasting with mouse in which the only 500 bp proximal sequence appeared to be sufficient. Another difference between rat and mouse was apparent in the 183 bp region of the rat promoter which induced a 3-fold stimulation of thymidine kinase promoter activity in both alphaT3-1 and CHO cells. Subsequent deletion analysis of the region residing between -1261 and -519 revealed the presence of multiple regulatory domains that contributed to the cell-specific activity. However, despite this efficiency in the context of the wild-type promoter, they failed to induce the activity of the minimal thymidine kinase (TK) promoter in the absence of the proximal 600 bp promoter region. Accordingly, a composite TK promoter containing the entire 1.2 kb promoter induced a 10-fold increase in the activity of the TK promoter in alphaT3-1 cells. Taken together these data suggest that distal regulatory regions are critical and require cooperation with proximal specific-promoter elements for activating basal R-GnRH gene expression in gonadotrope cells.
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PMID:Multiple elements in the distal part of the 1.2 kb 5'-flanking region of the rat GnRH receptor gene regulate gonadotrope-specific expression conferred by proximal domain. 986 30

The coding region of the human histone H4 gene FO108 undergoes dynamic changes in chromatin structure that correlate with modifications in gene expression. Such structural alterations generally reflect transcription factor interactions with gene regulatory sequences. To test for regulatory elements within the coding region, we performed transient transfection experiments in HeLa cells using constructs with histone H4 sequences fused upstream of a heterologous thymidine kinase promoter and CAT reporter gene. H4 gene sequences from -10 to +210 repressed transcription 4.8-fold. Further deletion and mutational analysis delineated three repressor elements within this region. Using oligonucleotide competition analysis and specific antibody recognition in electrophoretic mobility shift assays, as well as methylation interference and DNase I footprinting analyses, we have identified the CCAAT displacement protein (CDP/cut) as the factor that interacts with these three repressor elements. CDP/cut binding to these repressor sites is proliferation-specific and cell-cycle-regulated, increasing in mid to late S phase. Our results indicate that the proximal 200 nucleotides of the histone H4-coding region contain transcriptional regulatory elements that may contribute to cell-cycle control of histone gene expression by interacting with repressor complexes containing CDP/cut homeodomain transcription factors.
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PMID:Repressor elements in the coding region of the human histone H4 gene interact with the transcription factor CDP/cut. 987 97

The herpes simplex virus type 1 (HSV-1) thymidine kinase (TK) gene promoter contains binding sites for the cellular transcription factors such as Spl, CTF, and TFIID, each of which affects basal level expression of the TK gene. The transcription of the TK gene was induced by viral immediate early proteins, ICP0 and ICP4 in an additive manner, but was repressed by ICP22 and ICP27. To gain further insights into the role of ICP0 and ICP4 for expression of the TK gene during virus infection, several mutants with deletions or point mutations in each of the transcriptional regulatory elements were generated starting at -109 and progressing toward +1. According to the CAT assay involving these mutants, the cellular transcription factor (CTF) binding site was necessary for efficient expression in the presence of transfected ICP0 and ICP4 or during virus infection, whereas the Sp1 binding site had a minor effect on ICP0-mediated TK expression. These results indicate that the immediate early proteins of HSV-1 regulate expression of the TK gene during virus infection by modulating activities of cellular transcription factors such as CTF.
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PMID:Activation of the thymidine kinase promoter by herpes simplex virus type 1 immediate early proteins. 1042 Sep 86

The differentiation of nontransformed 3T3T mesenchymal stem cells is a multistep process that is associated with the progressive repression of mitogenic responsiveness to serum growth factors that ultimately results in expression of the terminally differentiated adipocyte phenotype. The repression of serum-induced mitogenesis by differentiation correlates with repression of the serum-inducible transcription of junB and c-fos. In contrast, the differentiation of neoplastically transformed cells does not repress mitogenic responsiveness or junB or c-fos inducibility. Because the junB and c-fos promoters both contain serum response elements (SREs), the current studies tested the possibility that differentiation might repress the ability of serum response factor (SRF) to bind to the SRE in normal cells but not in transformed cells. We now report that differentiation represses SRE serum inducibility using nontransformed cells transiently transfected with pjunB SRE thymidine kinase/chloroamphenicol acetyltransferase (SREtk/CAT) or pc-fos SREtk/CAT containing an intact SRF-binding domain. Adipocyte differentiation of nontransformed cells also markedly represses the ability of SRF to bind to the junB SRE, the c-fos SRE, and other SREs, as determined by mobility shift and gel supershift assays, without affecting the DNA binding characteristics of the nuclear protein SP-1. By comparison, the ability of SRF to bind SRE is not repressed by the differentiation of SV40 large T antigen-transformed 3T3T cells. The results further establish that adipocyte differentiation blocks the nuclear localization of SRF, thus preventing its interaction with SREs in nontransformed cells but not in transformed cells.
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PMID:Transformation blocks differentiation-induced inhibition of serum response factor interactions with serum response elements. 1044 98

We tested the virulence in mice of Toxoplasma gondii RH strain tachyzoites containing various copies of the chloramphenicol acetyl transferase-herpes simplex virus thymidine kinase fusion sequence (CAT-HSTK). Tachyzoite isolates containing >/=five copies of the fusion sequence were not lethal to female CD-1 outbred or BALB/c inbred mice, at doses up to 10(6) parasites, while the parental RH strain caused 100% mortality within 2 weeks at doses as low as 10 parasites. Mice infected with CTK11, an isolate containing five copies of the fusion sequence, showed no overt symptoms of disease and were protected from lethal challenge with the parental RH strain. The CTK11 isolate showed no difference in growth rate, the rate of host cell invasion, or extracellular viability in cell culture compared with parental RH parasites, demonstrating that the CAT-HSTK fusion protein does not affect the normal viability of this isolate. B11, B11C, and D1 isolates contained one or two copies of the CAT-HSTK coding sequence, were not sensitive to thymidine in cell culture, and caused 100% mortality in CD-1 outbred mice in <12 days. A fourth isolate, D1C, contained seven copies of the CAT-HSTK fusion sequence and was sensitive to exogenous thymidine (50% inhibitory concentration = 5.5 microM). Mice infected with D1C showed no symptoms of disease and survived beyond 90 days, thus correlating increased CAT-HSTK gene copies with thymidine sensitivity in cell culture and attenuated virulence in mice. BALB/c mice containing a targeted disruption of the gamma interferon gene (gko) were also susceptible to infection with CTK11 parasites but could be rescued by administration of subcutaneous thymidine once each day for 5 or 10 days following infection. These results suggest that the attenuation of CAT-HSTK(+) isolates in mice is directly due to active thymidine kinase that likely alters the pyrimidine biosynthetic pathway in these parasites.
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PMID:Expression of herpes simplex virus thymidine kinase in Toxoplasma gondii attenuates tachyzoite virulence in mice. 1049 8

We showed previously that microtubule disassembly by vinblastine induces the proto-oncogene c-myc in epithelial mammary HBL100 cells. In this study, we demonstrate that vinblastine treatment in these cells, in contrast to what was observed with the colon adenocarcinoma cell line HT29-D4, activated the transcription factor NFkappaB, which has been involved in c-myc regulation. The microtubule disassembly also induced IkappaB degradation. Using transient transfection analysis, we show that the trans-activation of c-myc by vinblastine was decreased when NFkappaB binding sites on c-myc promoter were mutated. Additionally, we demonstrate that microtubule dissolution trans-activated a thymidine kinase-CAT construct containing an NFkappaB binding site at -1180 to -1080 bp relative to the P1 promoter. Thus, vinblastine up-regulates the enhancer activity of the NFkappaB binding site. These results suggest that microtubule disassembly induced by vinblastine can trans-activate the c-myc oncogene through NFkappaB. Taking into consideration the paradoxical roles of both c-myc and NFkappaB in proliferation or apoptosis, this data reveals the complex action mechanism of this microtubule interfering agent.
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PMID:Involvement of nuclear factor kappaB in c-Myc induction by tubulin polymerization inhibitors. 1130

New and more potent progestins and antiprogestins suitable for reproductive therapy and contraception are currently the target of intensive research. The design of such drugs has been hampered by the complex technology required for screening these compounds at the molecular level. To solve this problem, we developed an in vitro cell system that allows detection of the progestagenic effects of a given compound using a PRE2-TATA-CAT reporter vector transiently introduced in a cell line stably transfected with the rabbit progesterone receptor (PR). The African Green Monkey Kidney CV1 (AGMK-CV1) cell line was chosen because these cells do not express endogenous steroid receptors; the selected clone stably expressing the rabbit PR has been maintained in our laboratory for more than 2 yr without detectable losses in PR content and progestagenic response. The presence and function of the PR were assessed by immunohistochemical and saturation analyses as well as by monitoring transactivation of the PRE2-TATA-CAT reporter gene. In this cell line, the PR is expressed at a concentration of 0.170 fmol/mg of protein, and the receptor is localized within the cell nucleus in either the presence or absence of the potent synthetic progestin R5020. This PR-expressing cell system allowed study of the in vitro progestational activity of several 19-nor progestins. The antiprogestin RU486 inhibited CAT activity induced by R5020; norethisterone (NET), levonorgestrel (LNG), and gestodene (GSD) induced PRE2-TATA-CAT activity at concentrations similar to those of R5020, whereas NET A-ring-reduced metabolites induced CAT activity at an extent lower than (5alpha-NET) or similar (3beta,5alpha-NET) to that of the precursor compound. The PRE2-TATA-CAT induction by 17beta-estradiol was also analyzed and no crossreactivity was detected. However, when the ERE-VitA2-TK-CAT (estrogen-responsive element-vitellogenin A2-thymidine kinase promoter-CAT) reporter vector and the estradiol receptor alpha or beta were cotransfected, CAT activity was induced in the presence of 17beta-estradiol, and NET tetrahydro-reduced derivatives. The results indicate that this AGMK-CV1-PR cell assay system appears to be suitable for measuring the effects of different synthetic progestins at the transcriptional level. In this assay system, NET, LNG, and GSD exhibit potent progestational effects at the transcriptional level. In the particular case of NET, the assay system allowed us to determine that the single or multiple hormonal transcriptional effects of this compound are partially mediated by its A-ring-reduced derivatives.
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PMID:Transactivation of progestin- and estrogen-responsive promoters by 19-nor progestins in African Green Monkey Kidney CV1 cells. 1195 66

We report the first mutational study of thymidine kinase 1 (TK1) performed in human solid tumors. We sequenced cDNAs representing the complete coding region of TK1 in human breast (n=22) and colorectal (n=26) cancer. Codon 106 near the ATP binding site constantly differed (ATG --> GTG; Met --> Val) from the one deposited by Bradshaw and Deininger in the Genbank database (Accession number NM_003258). Silent polymorphisms at codon 11 (CCC --> CCT; Pro --> Pro) and codon 75 (GCG --> GCA; Ala --> Ala) were frequently detected in tumors as well as in normal tissues. In breast cancer the two polymorphisms were observed in 63.6% of the samples analyzed. No significant association could be found between polymorphisms and TK activity. In colorectal cancer the incidence of the two changes was 73.1% and 69.2%, respectively. Interestingly, one colon cancer with high cytosolic TK activity displayed two missense mutations located in and near the putative phosphorylation site by tyrosine kinase (s) (TAT --> CAT; Tyr --> His) and by cAMP-, cGMP-dependent protein kinase (TAC --> TGC; Tyr --> Cys), respectively; adjacent normal mucosa showed no mutation. This may open new avenues that imply TK1 activity in tumor cell proliferation.
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PMID:Mutation analysis in the coding sequence of thymidine kinase 1 in breast and colorectal cancer. 1269 56

We replaced capsid genes by reporter genes and assessed expression in different types of human cancer cells and their normal counterparts, either at the level of whole cell population (CAT ELISA) or at the single cell level [FACS analysis of green fluorescent protein (GFP)]. CAT expression was substantial (up to 10,000 times background) in all infected tumor cells, despite variations according to the cell types. In contrast, no gene expression was detected in similarly infected normal cells (with t he exception of an expression slightly above background in fibroblasts). FACS analysis of GFP expression revealed that most tumor cells expressed high level of GFP while no GFP-positive normal cells could be detected with the exception of very few (less than 0.1%) human fibroblast cells expressing high level of GFP. We also replace capsid genes by genes coding for the costimulatory molecules B7-1 and B7-2 and show that, upon infection with B7 recombinant virions, only tumor cells display the costimulatory molecules and their immunogenicity was increased without any effect on normal cells. Using a recombinant minute virus of mice (MVM) containing the herpes simplex thymidine kinase gene, we could get efficient killing of most tumor cell types in the presence of ganciclovir. without affecting normal proliferating cells. The prospects and limitations of these different strategies are discussed.
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PMID:Oncoselective parvoviral vector-mediated gene therapy of cancer. 1272 35


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