Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:2.7.1.21 (thymidine kinase)
 7,561 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The potential of the CREM family of proteins to activate transcription of the genes encoding the testis-specific isozyme of angiotensin converting enzyme (ACET) and the gluconeogenic enzyme, phosphoenolpyruvate carboxykinase (GTP) (PEPCK) (EC 4.1.1.32) were investigated. Both CREM tau and CREM alpha bind efficiently to the putative cyclic AMP response element (CRE) present in the ACET gene (CRET) and to the CRE in the PEPCK gene. In HepG2 cells, the CRE was required for the strong stimulation by CREM tau of the expression of a chimeric PEPCK (-210 to +73)-chloramphenicol acetyl transferase (CAT) gene. The CRE could be mutated to the CRET sequence without losing the stimulatory effects of CREM tau. However, a similar chimeric gene driven by the regulatory region of the ACET gene, which contains the CRET site, could only be stimulated by CREM tau when its imperfect TATA element was mutated to an authentic TATA. Surprisingly, CREM alpha, an alleged inhibitor of CRE-mediated transcription, stimulated the expression of both PEPCK-CAT and ACET-CAT genes in HepG2 cells, a process which required the presence of the CRE and the CRET sites, respectively. In contrast, when the same CRE elements were used to drive the transcription of a chimeric gene containing the thymidine kinase promoter linked to the CAT structural gene, CREM alpha inhibited its expression in HepG2 and JEG3 cells. The expression of the same chimeric gene, however, was stimulated by CREM alpha in F9 embryonal carcinoma cells. These results demonstrated that the nature of the transcriptional effects of CREM isoforms on CRE-mediated transcription depends on the specific gene, the specific cell type and the promoter context of the CRE site.
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PMID:The cyclic AMP response elements of the genes for angiotensin converting enzyme and phosphoenolpyruvate carboxykinase (GTP) can mediate transcriptional activation by CREM tau and CREM alpha. 764 72

We observed that decline of thymidine kinase (TK) enzyme activity was severalfold faster than the decay of full length TK mRNA during growth arrest of 3T6 mouse fibroblasts or during differentiation of myoblasts (C2Cl12) or F9 embryonal carcinoma cells. In order to study the molecular mechanism of this disparate behavior, a polyclonal antiserum against mouse TK was raised in rabbit. High level expression of mouse TK polypeptide in Escherichia coli was achieved with a T7 RNA polymerase-directed expression system. Using the antiserum in immunoblotting, no indication for a pool of inactive enzyme was found during differentiation of F9 or growth arrest of 3T6 cells. Pulse labeling of these cells in vivo with [35S]methionine showed a more than 6-fold decrease in the rate of TK-protein synthesis of in F9 cells after 3 days of treatment with retinoic acid as well as in 3T6 cells after 16 h under low serum. This was not due to increased turnover of the protein as measured in pulse chase experiments. In addition, full length TK mRNA stayed associated with polysomes under these conditions in F9 as well as 3T6 cells. Taken together the results suggest that endogenous TK mRNA becomes translationally repressed under a variety of conditions when mouse cells cease to grow.
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PMID:Translational repression of endogenous thymidine kinase mRNA in differentiating and arresting mouse cells. 768 82

BOX DNA was previously isolated from the DNA sequence inserted in the enhancer B domain of mutant polyomavirus (fPyF9) DNA. We also reported that BOX DNA functioned negatively on DNA replication and transcription of another polyomavirus mutant (PyhrN2) in F9-28 cells, a subclone of mouse F9 embryonal carcinoma (EC) cells expressing the polyomavirus large T antigen. In this study, we demonstrate that BOX DNA enhances transcription from the thymidine kinase (TK) promoter in various EC cells. One or three copies of BOX DNA, linked to the bacterial chloramphenicol acetyltransferase gene under the control of the herpes simplex virus TK promoter, activated promoter activity in F9, P19, and ECA2 cells. Band shift assays using BOX DNA as a probe revealed that specific binding proteins were present in all EC cells examined; the patterns of BOX DNA-protein complexes were the same among them. A mutation introduced within BOX DNA abolished enhancer activity as well as the formation of specific DNA-protein complexes. In non-EC cells, including L and BALB/3T3 cells, the enhancer activity of BOX DNA on the TK promoter was not observed, although binding proteins specific to the sequence exist. In band shift assays, the patterns of the DNA-protein complexes of either L or BALB/3T3 cells were different from those of EC cells. Furthermore, the enhancer activity of BOX DNA decreased upon differentiation induction in all EC cells examined, of different origins and distinct differentiation ability. In parallel with the loss of enhancer activity, the binding proteins specific for BOX DNA decreased in these cells. Moreover, we cloned a genomic DNA of F9, termed BOXF1, containing BOX DNA sequence approximately 400 bp upstream from the RNA start site of the gene. BOXF1, containing a TATA-like motif and the binding elements for Sp1 and Oct in addition to BOX DNA, possessed promoter activity deduced by a BOXF1-chloramphenicol acetyltransferase construct. Deletion analyses of the construct revealed that the transcription of BOXF1 gene is regulated by BOX DNA, preferentially in undifferentiated EC cells versus differentiated cells. Hence, BOX DNA is probably a novel transcriptional element related to EC cell differentiation.
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PMID:BOX DNA: a novel regulatory element related to embryonal carcinoma cell differentiation. 790 32

This report demonstrates that the plasmids, pBLCAT2 and pBLCAT3, which are used widely for the preparation of promoter reporter gene constructs, exhibit cryptic promoter activity when expressed in embryonal carcinoma (EC) cells and their differentiated cells. The promoterless plasmid pBLCAT3 is used widely because it has two multiple cloning sites. We demonstrate that the activity of the cryptic promoter present in pBLCAT3 is increased dramatically by an enhancerlike region of the murine k-FGF gene. However, the basal cryptic promoter activity and the enhanced cryptic promoter activity can be silenced effectively by the insertion of three tandemly arranged polyadenylation sequences. To characterize the influence of the cryptic promoter in pBLCAT3, we tested its effects on two promoters. Our findings suggest that the cryptic promoter increases by several fold the expression of the reporter gene in pBLCAT2, which contains the thymidine kinase promoter. In contrast, the cryptic promoter present in pBLCAT3 does not seem to influence the expression of the k-FGF promoter. Last, we observed cryptic promoter activity when pBLCAT3 was expressed transiently in EC-differentiated cells. Together, our findings argue that transcription silencing sequences should be used when examining weak promoters in these plasmids, especially in combination with enhancers.
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PMID:Cryptic promoter activity within the backbone of a plasmid commonly used to prepare promoter/reporter gene constructs. 795 17

The relationship between DNA structure of replacement vectors and gene targeting efficiency was studied using positive-negative selection. The vectors contained pBR322 DNA, a bacterial neomycin-resistance gene (neo) for positive selection, a herpes simplex virus (HSV) thymidine kinase gene (tk) for negative selection, and a mouse genomic fragment, including exons 1 to 3 of the transthyretin (ttr) gene. The neo gene that confers G418 resistance was inserted into the second ttr exon, and the HSV-tk gene that confers gancyclovir (GANC) sensitivity was added to the 3' end of the ttr fragment. The vectors were linearized by digesting with restriction enzyme(s) and transfected into mouse embryonal carcinoma F9 cells. In this system, the enrichment by GANC selection as well as the frequency of gene targeting was increased by placing the pBR322 DNA at the 3' end of the HSV-tk gene. Adding one more HSV-tk gene at the 5' end of the ttr fragment did not increase the enrichment by GANC selection. This enrichment factor was also increased by reducing the size of the ttr fragment present between the two selection markers. However, it decreased the frequency of gene targeting and, overall, it did not increase the efficiency of isolating targeted clones. When structures of the vector DNA fragments present in 20 G418-resistant and GANC-resistant non-targeted clones were examined by Southern blot analysis, the inefficiency of GANC selection proved to be mostly caused by exonucleolytic degradation of HSV-tk genes progressing from ends of the vectors.
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PMID:Structures of replacement vectors for efficient gene targeting. 805 60

We reported that a cell surface thrombin receptor, thrombomodulin (TM), was regulated by cyclic AMP in fibroblasts and in parietal endoderm-like cells derived from F9 embryonal carcinoma cells. In this paper, the genetic basis for augmentation of TM expression by cyclic AMP was studied in F9 and BALB/3T3 cells. Transient expression assays were performed with plasmid constructs containing various 5' flanking sequences of the TM gene and a reporter gene, chloramphenicol acetyltransferase (CAT). Two regulatory DNA regions, the proximal (-411 to -50) and the distal (-1026 to -850), were located. Interplay of the two regions was suggested using a heterologous thymidine kinase promoter in differentiated F9 cells. Both proximal and distal regions contributed to cyclic AMP-dependent augmentation of CAT expression in differentiated F9 cells, whereas only the proximal region was functional in BALB/3T3 cells. The two cell types responded differently also to a protein synthesis inhibitor, cycloheximide, with respect to TM message accumulation. In BALB/3T3 cells TM message accumulation was refractory to the inhibitor in contrast to that of differentiated F9 cells, which was only partially so. We propose that there are at least two separate genomic DNA regions that regulate cyclic AMP-dependent TM gene expression and that their functions are cell type dependent.
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PMID:Cyclic AMP-mediated augmentation of thrombomodulin gene expression: cell type-dependent usage of control regions. 839 1

A replacement vector convenient for introducing subtle mutations into various mouse genes has been developed using, a model system, the mouse transthyretin-encoding gene (ttr) and mouse embryonal carcinoma F9 cells. The vector consists of part of ttr carrying a subtle mutation in its second exon, and a cassette of the neomycin-resistance (neo)- and herpes simplex virus thymidine kinase (HSV-tk)-encoding genes flanked with a 3-kb duplication of mostly the second intron of ttr. In the first step ('replacement'), part of the endogenous ttr was replaced by vector DNA via homologous recombination, and two such clones, #33 and #77, were isolated from 185 G418-resistant clones by allele-specific PCR. In the second step ('excision'), gancyclovir-resistant colonies were screened, and 7 and 84% of those isolated from clones #33 and #77, respectively, were demonstrated to carry the subtle mutation in ttr, without the cassette of selection markers. In five independently isolated random integrants of the same vector DNA, the cassette of selection markers was excised efficiently by recombination within the duplication.
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PMID:A replacement vector used to introduce subtle mutations into mouse genes. 854 62

It is important to develop a system to express therapeutic genes in tumor cells with sufficient selectivity for cancer gene therapy. Midkine (MK) is a newly identified heparin-binding growth factor that is transiently expressed in the early stages of retinoic acid-induced differentiation of embryonal carcinoma cells. It has been reported that many human malignant tumors express high levels of MK mRNA or protein. However, no MK expression is detected in human or mouse liver. These interesting features of MK led us to examine the MK promoter as a candidate for tumor-specific gene expression. We thus developed new recombinant adenoviral (Ad) vectors containing either luciferase reporter gene (AdMKLuc) or herpes simplex thymidine kinase gene (AdMKTK) under the control of the human MK promoter. AdMKLuc achieved relatively high activity in Wilms' tumor (G-401) and neuroblastoma (SK-N-SH) cell lines. In addition, AdMKTK induced marked cell death in response to ganciclovir (GCV) in these same lines. Conversely, very low activity of the MK promoter was observed in mouse liver in vivo compared with the cytomegalovirus promoter. Importantly, AdMKTK + GCV did not induce liver toxicity, whereas substantial toxicity was seen with AdCMVTK + GCV treatment. On the basis of these findings, we conclude that the MK promoter is a candidate tumor-specific promoter for Wilms' tumor or neuroblastoma.
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PMID:Midkine promoter-based adenoviral vector gene delivery for pediatric solid tumors. 1096 65


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