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)

A series of replication-defective retroviral vectors were assessed for their ability to efficiently transfer functional genes into undifferentiated cells. In these vectors (designated handicapped because of a deletion of enhancer and promoter sequences in the viral long terminal repeat) transcription of inserted genes is under the control of internal promoters. Although a composite promoter composed of a mutant polyoma virus enhancer (PyF441) coupled to the herpes simplex virus thymidine kinase promoter was anticipated to function efficiently, it was found to be significantly inferior to the mouse X-chromosome phosphoglycerate kinase (pgk-1) promoter, in its ability to express the selectable neomycin phosphotransferase gene in mouse embryonal carcinoma cells. The pHMB vector, which contains the pgk-1 promoter, was shown to confer the drug-resistant phenotype at high frequencies to F9 and P19 cells. This vector might prove to be of general utility for efficient gene expression in other developmental contexts.
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PMID:Comparative analysis of retroviral vector expression in mouse embryonal carcinoma cells. 256 Feb 17

Maloney murine leukemia virus-based, replication-defective retroviral vectors containing the neomycin resistance gene (neo) were developed to transfer the Escherichia coli ada gene coding for O6-alkylguanine-DNA alkyltransferase, into mammalian cells. To optimize gene transfer and expression, the following promoters were linked to ada: the Maloney murine leukemia virus promoter within the long-terminal repeat, the Rous sarcoma virus promoter, the thymidine kinase promoter, or the human phosphoglycerate kinase promoter. Sequences were transfected into the helper virus-free retroviral packaging psi-2 cell line. Recombinant retroviruses were tested in CCL-1 cells, which, like most murine tissues, have low levels of alkyltransferase and are sensitive to 1,3-bis(2-chloroethyl)nitrosourea (BCNU), and in NIH-3T3 cells, which are BCNU resistant and have high levels of alkyltransferase. Lines infected with each of the four retroviruses were selected for neo expression and found to have intact proviral integration and ada gene expression. Alkyltransferase activity was greatest with retrovirus containing the Rous sarcoma virus-ada gene; infected NIH-3T3 cells had up to 2300 units of alkyltransferase/mg of protein compared with 151 units/mg of protein in control cells, and infected CCL-1 cells had up to 1231 units/mg of protein compared with 33 units/mg of protein in control cells. CCL-1 cells expressing ada were more resistant to BCNU cytotoxicity than were controls. However, NIH-3T3 cells expressing ada were only slightly more resistant to BCNU than controls, possibly because most of the ada protein was cytoplasmic rather than nuclear as suggested by immunohistochemical stain. These studies establish a series of retroviruses containing the bacterial ada gene, which efficiently infect mammalian cells. ada expression increases nitrosourea resistance in cells with low mammalian alkyltransferase activity.
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PMID:Increase in nitrosourea resistance in mammalian cells by retrovirally mediated gene transfer of bacterial O6-alkylguanine-DNA alkyltransferase. 267 54

Chromosome-mediated gene transfer (CMGT) of the human genes for hypoxanthine phosphoribosyl transferase (HPRT) and cytosol thymidine kinase (TK1) into HPRT deficient mouse A9 cells or TK deficient Swiss mouse 3T3TK- cells was found to occur at frequencies at least one order of magnitude higher than DNA-mediated gene transfer (DMGT). The frequency of CMGT into 3T3TK- cells was reduced by more than an order of magnitude by a posttreatment of the recipient cells with dimethyl sulphoxide (DMSO). After CMGT, expression of the non-selected genes coding for galactokinase (GALK) and acid alpha-glucosidase (GAA), both syntenic with TK1, was observed in a number of transformants. From the pattern of cotransfer, a tentative gene ordering of CENTROMERE-GALK-TK1-GAA on human chromosome 17 was deduced. Chromosome-mediated cotransfer of X-linked human phosphoglycerate kinase (PGK) with HPRT was observed in two out of 33 A9 transformants analysed. DNA-mediated cotransfer of a syntenic gene was only observed for GALK, cotransferred with TK1 in two out of 18 TK+ transformants of mouse LTK- cells. Therefore, with murine cells as recipients of human donor genetic material, CMGT results in a higher frequency of transfer and a higher incidence of cotransfer of syntenic genes than DMGT using cellular DNA in the same cell system.
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PMID:Cotransfer of syntenic human genes into mouse cells using isolated metaphase chromosomes or cellular DNA. 388 35

Chicken glyceraldehyde-3-phosphate dehydrogenase gene (GAPD) and thymidine kinase gene (TK) were co-transfected into mouse LMTK- cells by the calcium phosphate precipitation technique. Four of the eight hypoxanthine/aminopterin/thymidine-containing medium-resistant, TK+ transfectants were shown to produce different amounts of chicken glyceraldehyde-3-phosphate dehydrogenase by zymogram analysis. Subcloning and further analysis revealed that the chicken GAPD was stably inherited and that its enzyme subunits randomly combined with mouse subunits in heterotetramers. Although the contribution of chicken enzyme varied from approximately 30 to approximately 90% of the total glyceraldehyde-3-phosphate dehydrogenase activity with a proportional increase in total activity in the different subclones, it did not appear to affect the expression of mouse endogenous glycolytic enzymes since there was no distinct change in the levels of either mouse glyceraldehyde-3-phosphate dehydrogenase mRNA nor mouse phosphoglycerate kinase enzyme activity. The levels of chicken GAPD copy number, mRNA, and enzyme apparently were generally correlated in the different subclones, suggesting that the chicken GAPD in the mouse cells were expressed constitutively. In situ hybridization revealed that the transfected genes were integrated into mouse chromosomes in one cluster, and the locations of these clusters were different in different clones. Chromatin structure analyses of the chicken GAPD in four different transfectants revealed three DNase I-hypersensitive sites located around 0.2, 2.0, and 3.4 kilobases upstream from the 5' side of the gene. These sites are also present in the same locations in chicken lymphoblastoid cells (Kuo, M. T., Iyer, B., and Schwartz, R. J. (1982) Nucleic Acids Res 10, 4565-4579), indicating the dominant transmission of DNase I-hypersensitive cleavage sites in the transfected gene.
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PMID:The expression and chromatin structure of the chicken glyceraldehyde-3-phosphate dehydrogenase gene in mouse cells. 397 17

The introduction of small mutations instead of null alleles into the mouse genome has broad applications to the study of protein structure-function relationships and the creation of animal models of human genetic diseases. To test a simple mutational strategy we designed a targeting vector for the mouse proopiomelanocortin (POMC) gene containing a single nucleotide insertion that converts the initial tyrosine codon of beta-endorphin 1-31 to a premature translational termination codon and introduces a unique Hpal endonuclease restriction site. The targeting vector also contains a neo cassette immediately 3' to the last POMC exon and a herpes simplex virus thymidine kinase cassette to allow positive and negative selection. Homologous recombination occurred at a frequency of 1/30 clones of electroporated embryonic stem cells selected in G418 and gancyclovir. 10/11 clones identified initially by a polymerase chain reaction (PCR) strategy had the predicted structure without evidence of concatemer formation by Southern blot analysis. We used a combination of Hpa I digestion of PCR amplified fragments and direct nucleotide sequencing to further confirm that the point mutation was retained in 9/10 clones. The POMC gene was transcriptionally silent in embryonic stem cells and the targeted allele was not activated by the downstream phosphoglycerate kinase-1 promoter that transcribed the neo gene. Under the electroporation conditions used, we have demonstrated that a point mutation can be introduced with high efficiency and precision into the POMC gene using a replacement type vector containing a retained selectable marker without affecting expression of the allele in the embryonic stem cells. A similar strategy may be useful for a wide range of genes.
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PMID:Introduction of a point mutation into the mouse genome by homologous recombination in embryonic stem cells using a replacement type vector with a selectable marker. 839 2

The glycolytic enzyme phosphoglycerate kinase (PGK) consists of two isozymes, somatic-type PGK-1 and testis-specific PGK-2. The isozyme switch from PGK-1 to PGK-2 occurs during spermatogenesis at the mRNA level. The distal upstream region of the gene encoding mouse PGK-2 (Pgk-2) possesses a silencer-like negative cis element. In the present study, a positive cis element located in the proximal upstream region and factor(s) bound to it were analyzed in vitro. Cell-free transcription using nuclear extracts of rat organs demonstrated that the region between nucleotide positions -82 and -64, relative to the most distal transcription initiation site at +1, stimulates transcription in testis extracts. The cis element did not act on the promoter of the thymidine kinase gene, suggesting that it stimulates Pgk-2 transcription in a promoter-specific manner. The cis element bound a nuclear factor(s), which we designated TAP-1. Introducing various base substitutions within the cis element revealed that TAP-1-binding to the element requires the sequence 5'-GGAA-3', which is the binding motif for Ets oncoproteins. We previously reported that TIN-1, a transcription inhibitor of Pgk-1, binds to a sequence similar to the Ets-binding site. The addition of an oligo DNA containing the TIN-1-binding sequence of Pgk-1 prevented TAP-1 from binding to the Pgk-2 cis element, and vice versa. These results suggest that both TIN-1 and TAP-1, which are presumably involved in transcription regulation of the two Pgk genes, recognize DNA sequences related to the Ets-binding motif.
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PMID:A factor stimulating transcription of the testis-specific Pgk-2 gene recognizes a sequence similar to the binding site for a transcription inhibitor of the somatic-type Pgk-1 gene. 844 29

In recent years the idea of using gene therapy as a modality in the treatment of diseases other than genetically inherited, monogenic disorders has taken root. This is particularly obvious in the field of oncology where currently more than 100 clinical trials have been approved worldwide. This report will summarize some of the exciting progress that has recently been made with respect to both targeting the delivery of potentially therapeutic genes to tumor sites and regulating their expression within the tumor microenvironment. In order to specifically target malignant cells while at the same time sparing normal tissue, cancer gene therapy will need to combine highly selective gene delivery with highly specific gene expression, specific gene product activity, and, possibly, specific drug activation. Although the efficient delivery of DNA to tumor sites remains a formidable task, progress has been made in recent years using both viral (retrovirus, adenovirus, adeno-associated virus) and nonviral (liposomes, gene gun, injection) methods. In this report emphasis will be placed on targeted rather than high-efficiency delivery, although those would need to be combined in the future for effective therapy. To date delivery has been targeted to tumor-specific and tissue-specific antigens, such as epithelial growth factor receptor, c-kit receptor, and folate receptor, and these will be described in some detail. To increase specificity and safety of gene therapy further, the expression of the therapeutic gene needs to be tightly controlled within the target tissue. Targeted gene expression has been analyzed using tissue-specific promoters (breast-, prostate-, and melanoma-specific promoters) and disease-specific promoters (carcinoembryonic antigen, HER-2/neu, Myc-Max response elements, DF3/MUC). Alternatively, expression could be regulated externally with the use of radiation-induced promoters or tetracycline-responsive elements. Another novel possibility that will be discussed is the regulation of therapeutic gene products by tumor-specific gene splicing. Gene expression could also be targeted at conditions specific to the tumor microenvironment, such as glucose deprivation and hypoxia. We have concentrated on hypoxia-targeted gene expression and this report will discuss our progress in detail. Chronic hypoxia occurs in tissue that is more than 100-200 microns away from a functional blood supply. In solid tumors hypoxia is widespread both because cancer cells are more prolific than the invading endothelial cells that make up the blood vessels and because the newly formed blood supply is disorganized. Measurements of oxygen partial pressure in patients' tumors showed a high percentage of severe hypoxia readings (less than 2.5 mmHg), readings not seen in normal tissue. This is a major problem in the treatment of cancer, because hypoxic cells are resistant to radiotherapy and often to chemotherapy. However, severe hypoxia is also a physiological condition specific to tumors, which makes it a potentially exploitable target. We have utilized hypoxia response elements (HRE) derived from the oxygen-regulated phosphoglycerate kinase gene to control gene expression in human tumor cells in vitro and in experimental tumors. The list of genes that have been considered for use in the treatment of cancer is extensive. It includes cytokines and costimulatory cell surface molecules intended to induce an effective systemic immune response against tumor antigens that would not otherwise develop. Other inventive strategies include the use of internally expressed antibodies to target oncogenic proteins (intrabodies) and the use of antisense technology (antisense oligonucleotides, antigenes, and ribozymes). This report will concentrate more on novel genes encoding prodrug activating enzymes, so-called suicide genes (Herpes simplex virus thymidine kinase, Escherichia coli nitroreductase, E. (ABSTRACT TRUNCATED)
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PMID:Targeting gene therapy to cancer: a review. 940 37

The Cre-loxP recombination system of bacteriophage P1 is frequently utilized in genetic manipulation in embryonic stem (ES) cells. The level of Cre expression is critical to induce loxP site-specific recombination in ES cells. To compare the efficiency of recombination, we constructed four Cre expression vectors driven by different promoters: cytomegarovirus/chicken beta-actin (CAG) promoter, human polypeptide chain elongation factor 1alpha (hEF-1alpha) promoter, mouse phosphoglycerate kinase-1 (mPGK) promoter, and polyoma enhancer/herpes simplex virus thymidine kinase (MC1) promoter. We introduced these Cre expression vectors by electroporation into three ES cell lines carrying a single copy of CAG-loxP-chloramphenicol acetyltransferase (CAT) gene-loxP-beta-galactosidase (beta-gal) gene construct. Since the Cre-mediated recombination leads to excision of the CAT gene, the efficiency of recombination can be monitored as beta-gal expression. No selection system was used in the experiments. The maximum recombination frequency was obtained when the CAG promoter was used, followed by the hEF-1alpha promoter, the mPGK promoter and the MC1 promoter in order. These results indicate that the efficiency of recombination in transient expression system correlates with the promoter activity of Cre expression vector. Thus, it is important to choose the promoter for effective recombination by Cre.
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PMID:Efficiency of recombination by Cre transient expression in embryonic stem cells: comparison of various promoters. 944 13

The aim of this study was to examine whether the human alpha-fetoprotein (AFP) enhancer could be used to induce hepatocellular carcinoma (HCC)-selective expression of the herpes simplex virus thymidine kinase (HSV-tk) gene which is under the control of the phosphoglycerate kinase (pgk) promotor. The human AFP enhancer was linked with the non-tissue-specific, human housekeeping pgk promoter in a retroviral vector. AFP-producing HCC cells infected with retroviruses carrying the HSV-tk gene under the control of the AFP enhancer/pgk promoter were much more susceptible to the prodrug, ganciclovir (GCV), than those infected with the same retroviruses without the AFP enhancer. Non-HCC cells infected with retroviruses carrying the HSV-tk gene under the control of the AFP enhancer/pgk promoter exhibited profoundly increased resistance to GCV compared with those infected with the same retroviruses without the AFP enhancer. Northern blot analysis revealed that the AFP enhancer caused enhanced HSV-tk expression in AFP-producing HCC cells and suppressed HSV-tk expression in non-HCC cells. Our results indicate that the AFP enhancer could give HCC selectivity to the pgk promoter, and that this novel strategy may be useful for HCC-selective cancer gene therapy.
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PMID:Gene therapy for hepatocellular carcinoma based on tumour-selective suicide gene expression using the alpha-fetoprotein (AFP) enhancer and a housekeeping gene promoter. 1116 41

Gene therapy requires the presence of a robust and yet small promoter to drive high-level expression of desired proteins. In comparative analysis, we investigated the promoter strength of the CTP:phosphocholine cytidylyltransferase promoter (CCT alpha) with other commonly used promoters, which were all cloned into a similar background vector (PGL3 basic). Transient promoter-reporter assays in murine lung epithelial (MLE-12) cells revealed that the core CCT alpha promoter (240 bp) was observed to exhibit a 40-fold, 8-fold, and 3-fold higher level of activity compared with the simian virus 40, human cytomegalovirus, and Rous sarcoma virus promoters, respectively. The CCT alpha promoter was significantly more active than the Clara cell 10, thymidine kinase, and phosphoglycerate kinase promoters. This pattern of high-level expression for CCT alpha was detected primarily in cell lines of distal lung epithelial origin (MLE-12, RLE, H441) and was reduced in other cell lines (A549, CHO, HepG 2). CCT alpha promoter-reporter activity, CCT alpha transcript levels, and immunoreactive protein levels increased significantly in the presence of all-trans retinoic acid. The CCT alpha promoter, in a retinoic acid-inducible manner, efficiently directed expression of murine erythropoietin in MLE-12 cells. Collectively, these observations suggest that the CCT alpha construct might be useful to drive high-level, regulatable expression of heterologous proteins in alveolar epithelia.
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PMID:The CCT promoter directs high-level transgene expression in distal lung epithelial cell lines. 1282 50


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