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)

We have examined the expression of chimeric plasmids containing coding sequences for the herpes simplex virus thymidine kinase (tk) gene or the Tn5 gene for neomycin resistance (neo) linked to the late promoter of polyoma DNA. Although polyoma late genes are generally not expressed in transformed cells containing only integrated viral DNA molecules, rat tk- or wild-type cells transfected with the tk- or neo-containing plasmids were capable of growing in medium containing either hypoxanthine-aminopterin-thymidine or G418, respectively, under conditions nonpermissive for extrachromosomal DNA replication, indicating that the tk or neo genes were fully expressed. Moreover, cells were capable of growth in either hypoxanthine-aminopterin-thymidine or G418, even in the absence of direct selection for this activity. Northern analysis indicated steady-state levels of tk or neo transcripts that approximated the levels of polyoma early transcripts. S1 analysis showed that these transcripts initiated within the late promoter of polyoma and that their 5' ends mapped at positions similar or identical to those utilized during late lytic infection. The effect of substitution of polyadenylation signals was examined. Although plasmids containing the polyoma early polyadenylation signal were more efficient in conferring to cells a stable G418-resistant phenotype than similar constructions using the late signal, both signals were found to be effectively utilized. This indicates that the inability to detect late transcripts in polyoma-transformed cells in the absence of free viral DNA production is not an effect of inefficient mRNA cleavage or polyadenylation. Our results suggest that late gene expression in integrated polyoma genomes is not regulated at the level of message initiation but, most likely, through posttranscriptional events.
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PMID:Transcription from the polyoma late promoter in cells stably transformed by chimeric plasmids. 298 70

Retrovirus vectors offer a simple and highly efficient method for introducing new genes into mammalian cells. Here, we have examined the efficiency of gene transfer into hematopoietic cells with retrovirus vectors carrying the neomycin (neo) resistance gene expressed from different transcriptional regulatory regions. Direct infection of mouse bone marrow cells resulted in high efficiencies of gene transfer into a variety of myeloid progenitor cells, including pluripotent, erythroid, and granulocyte-macrophage colony-forming cells with all the vectors examined. However, the progeny derived from individual pluripotent progenitor cells infected with different vectors differed markedly in the proportion of G418-resistant progenitor cells, as judged by their ability to survive selection in the drug G418. This biological assay suggests that the highest level of expression was observed when the neo gene was expressed from constructs that contained the herpes thymidine kinase promoter rather than the viral long terminal repeat or the simian virus 40 early region promoter. In contrast, neo gene expression was highest in fibroblasts infected with the vector containing the simian virus 40 early region promoter. These results show that high and sustainable levels of gene expression in hematopoietic cells can be obtained with retrovirus vectors containing appropriate transcriptional regulatory regions.
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PMID:Modulation of gene expression in multiple hematopoietic cell lineages following retroviral vector gene transfer. 302 4

We have developed a system for studying spontaneous mutations at a chromosomally located single-copy HSV-1 thymidine kinase (TK) gene in the human 143 TK- cell line. The neo gene, which confers resistance to the antibiotic G418, was placed next to the TK gene for the purpose of screening out gross chromosomal alterations. TK- mutations were selected using the anti-TK nucleotide analogs trifluorothymidine, acyclovir, and DHPG 9-(1,3 dihydroxy-2-propoxymethyl)-guanine either separately, or in combination to eliminate leaky mutations. Analysis of the TK- mutations by Southern blotting revealed that the majority had undetectable alterations of less than 50 base pairs. The results using the methylation-sensitive enzymes HpaII, AvaI, and SmaI suggest that the inactivation of the TK gene was not due to extensive methylation, although specific methylation of a limited number of MspI sites cannot be ruled out. Reversion studies, however, showed that of 16 mutants analyzed, about half had a very high reversion frequency (approximately 10(-2). This suggests that inactivation of the TK gene may have occurred by a variety of mutational events.
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PMID:Analysis of spontaneous mutations in a chromosomally located HSV-1 thymidine kinase (TK) gene in a human cell line. 302

The liver is an important target for potential gene therapy because of the critical role it plays in intermediary metabolism and synthesis of serum proteins. We report the use of retroviral vectors for transfer of recombinant genes into primary mouse hepatocytes. Hepatocytes were grown in a defined serum-free medium and expressed liver-specific functions for up to 14 days. Hepatocytes were transformed to Genticin (G418) resistance by infection with recombinant retroviruses carrying the Tn5 neomycin-resistance gene. The G418-resistant cells exhibited characteristic hepatocyte morphology and continued to express liver-specific gene function. A retrovirus that expresses neomycin resistance driven by a herpes simplex thymidine kinase promoter produced the most efficient transformation compared with viruses using the retroviral long terminal repeat promoter or the simian virus 40 early-region promoter. These experiments indicate that primary hepatocytes can be successfully cultured and transformed with recombinant genes using retroviral vectors. These results provide a model for future somatic gene replacement therapy in which functional genes can be introduced into hepatocytes by viral-mediated gene transfer.
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PMID:Retroviral gene transfer into primary hepatocytes: implications for genetic therapy of liver-specific functions. 347 59

We have used a producer NIH 3T3 cell line that secretes, together with the helper Moloney murine leukemia virus (Mo-MuLV), a transducing recombinant virus containing the neomycin-resistance gene linked to the Mo-MuLV long terminal repeat (LTR). By infecting three embryonal carcinoma cell lines, PCC4.aza1R, F9tk-, and Nulli-SCC1, with this recombinant virus, we have isolated many transductant clones that stably express the integrated neomycin-resistance gene. These clonal transductant lines consist of undifferentiated embryonal carcinoma cells as judged by morphology, tumorigenicity in 129/Sv mice, and cell-surface antigenic markers. Analysis of the integrated recombinant viral genes by Southern blot hybridization revealed that some of the lines have single copies, whereas others have multiple copies, probably in multiple sites. Although these transductant lines contained many copies of helper Mo-MuLV integrated in the cellular genome, expression of these helper viruses was not detected either by reverse transcriptase activity or by X-C plaque assay. Two F9tk--derived, G418-resistant transductant lines were superinfected with a second recombinant transducing virus that contains the herpes simplex virus thymidine kinase gene flanked by the Mo-MuLV LTR. The frequency of transduction to yield clones able to grow in hypoxanthine/aminopterin/thymidine medium was similar to that of the parental F9tk- cells. These results suggest that the expression of the neomycin-resistance gene, linked to MoMuLV LTR in the transductant embryonal carcinoma cell clones, is due to a cisacting mechanism(s).
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PMID:Isolation of embryonal carcinoma cell lines that express integrated recombinant genes flanked by the Moloney murine leukemia virus long terminal repeat. 385 93

We have constructed a recombinant simian virus 40 (SV40) DNA containing a copy of the Harvey murine sarcoma virus long terminal repeat (LTR). This recombinant viral DNA was converted into an infectious SV40 virus particle and subsequently infected into NIH 3T3 cells (either uninfected or previously infected with Moloney leukemia virus). We found that this hybrid virus, SVLTR1, transforms cells with 10 to 20 times the efficiency of SV40 wild type. Southern blot analysis of these transformed cell genomic DNAs revealed that simple integration of the viral DNA within the retrovirus LTR cannot account for the enhanced transformation of the recombinant virus. A restriction fragment derived from the SVLTR-1 virus which contains an intact LTR was readily identified in a majority of the transformed cell DNAs. These results suggest that the LTR fragment which contains the attachment sites and flanking sequences for the proviral DNA duplex may be insufficient by itself to facilitate correct retrovirus integration and that some other functional element of the LTR is responsible for the increased transformation potential of this virus. We have found that a complete copy of the Harvey murine sarcoma virus LTR linked to well-defined structural genes lacking their own promoters (SV40 early region, thymidine kinase, and G418 resistance) can be effectively used to promote marker gene expression. To determine which element of the LTR served to enhance the biological activity of the recombinant virus described above, we deleted DNA sequences essential for promoter activity within the LTR. SV40 virus stocks reconstructed with this mutated copy of the Harvey murine sarcoma virus LTR still transform mouse cells at an enhanced frequency. We speculate that when the LTR is placed more than 1.5 kilobases from the SV40 early promoter, the cis-acting enhancer element within the LTR can increase the ability of the SV40 promoter to effectively operate when integrated in a murine chromosome. These data are discussed in terms of the apparent cell specificity of viral enhancer elements.
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PMID:Enhanced transformation by a simian virus 40 recombinant virus containing a Harvey murine sarcoma virus long terminal repeat. 630 72

A recombinant plasmid was constructed (pV69) which comprises a subgenomic fragment of bovine papilloma virus type 1 (BPV1) DNA, part of plasmid pBR322 DNA and a drug resistance gene expressed in both mammalian fibroblasts and Escherichia coli. This gene (vv2) is a modified form of the bacterial neomycin resistance gene (neo) linked to the herpes simplex virus thymidine kinase (tk) promoter (plasmid pAG60), to which the original bacterial neo promoter from transposon Tn5 was added back, upstream of the eukaryotic promoter. It induced kanamycin resistance in E. coli, as well as resistance to the drug G418 in rat and mouse fibroblasts. Its expression in FR3T3 rat cells was enhanced as compared with the original tk-neo construction. After transfer of plasmid pV69 into C127 mouse cells or FR3T3 rat cells, the number of resistant colonies selected in medium containing G418 was one to two orders of magnitude higher than that of transformed foci in normal medium. In eight independent cell lines selected by drug resistance, pV69 DNA was found to be maintained in a plasmidial state, without any detectable rearrangement or deletion and could be transferred back in E. coli. In contrast, cell lines selected by focus formation in normal medium maintained deleted forms of the original plasmid DNA, and only part of them were resistant to G418. Most of the drug-resistant clones had kept the morphology and growth control of the normal fibroblasts. However, with further passages in culture, these cells spontaneously produced transformed foci with increasing frequencies.
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PMID:Plasmidial maintenance in rodent fibroblasts of a BPV1-pBR322 shuttle vector without immediately apparent oncogenic transformation of the recipient cells. 632 68

Recombinant adeno-associated virus 2 (AAV) virions were constructed containing a gene for resistance to neomycin (neoR), under the control of either the herpesvirus thymidine kinase (TK) gene promoter (vTK-Neo), or the human parvovirus B19 p6 promoter (vB19-Neo), as well as those containing an upstream erythroid cell-specific enhancer (HS-2) from the locus control region of the human beta-globin gene cluster (vHS2-TK-Neo; vHS2-B19-Neo). These recombinant virions were used to infect either low density or highly enriched populations of CD34+ cells isolated from human umbilical cord blood. In clonogenic assays initiated with cells infected with the different recombinant AAV-Neo virions, equivalent high frequency transduction of the neoR gene into slow-cycling multipotential, erythroid, and granulocyte/macrophage (GM) progenitor cells, including those with high proliferative potential, was obtained without prestimulation with growth factors, indicating that these immature and mature hematopoietic progenitor cells were susceptible to infection by the recombinant AAV virions. Successful transduction did not require and was not enhanced by prestimulation of these cell populations with cytokines. The functional activity of the transduced neo gene was evident by the development of resistance to the drug G418, a neomycin analogue. Individual high and low proliferative colony-forming unit (CFU)-GM, burst-forming unit-erythroid, and CFU-granulocyte erythroid macrophage megakaryocyte colonies from mock-infected, or the recombinant virus-infected cultures were subjected to polymerase chain reaction analysis using a neo-specific synthetic oligonucleotide primer pair. A 276-bp DNA fragment that hybridized with a neo-specific DNA probe on Southern blots was only detected in those colonies cloned from the recombinant virus-infected cells, indicating stable integration of the transduced neo gene. These studies suggest that parvovirus-based vectors may prove to be a useful alternative to the more commonly used retroviral vectors for high efficiency gene transfer into slow or noncycling primitive hematopoietic progenitor cells, without the need for growth factor stimulation, which could potentially lead to differentiation of these cells before transplantation.
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PMID:Adeno-associated virus 2-mediated high efficiency gene transfer into immature and mature subsets of hematopoietic progenitor cells in human umbilical cord blood. 751 1

We previously demonstrated stable integration of a transduced thymidine kinase (TK)-neo gene into immature and replatable stem and progenitor cells, as assessed by the presence of the gene in second-generation colonies. To evaluate whether this integration was still present in third- and fourth-generation colonies, nonadherent low-density T-lymphocyte-depleted (NALT-) cells from human umbilical cord blood were prestimulated with recombinant human (rhu) erythropoietin (Epo), steel factor (SLF), interleukin-3 (IL-3), granulocyte-macrophage (GM) colony-stimulating factor (CSF), and granulocyte (G)-CSF. Prestimulated NALT- cells were incubated with retroviral-containing supernatant obtained from TK-neo vector-producing cells, washed, and assayed for colony formation in the presence of Epo, SLF, IL-3, GM-CSF, and G-CSF -/+ G418. The results confirmed that the TK-neo gene could be efficiently introduced into hematopoietic progenitor cells without stromal cells as a source of virus. As previously reported, proviral integration was detected in primary G418R-colonies, and in second-generation replated colonies derived from G418R granulocyte erythroid macrophage megakaryocyte colony-forming units and high-proliferative potential colony-forming cells (HPP-CFCs). Moreover, we now document that proviral integration was apparent in cells from colonies derived from third- and fourth-generation replated HPP-CFC, suggesting a high degree of stable integration of the transduced gene.
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PMID:Stable integration of retrovirally transduced genes into human umbilical cord blood high-proliferative potential colony-forming cells (HPP-CFC) as assessed after multiple HPP-CFC colony replatings in vitro. 774 19

A retroviral vector carrying both forward (neo) and backward (herpes simplex virus thymidine kinase or HSV-TK gene) selection markers was constructed as a substrate for mutational assay in mammalian cells. The cells infected with this virus are first selected with G418, mutagenized and then selected with the anti-herpes drug acyclovir (ACV). Since HSV-TK, but not the host TK, is capable of converting ACV to a toxic metabolite, cells retaining the intact HSV-TK gene fail to survive, while the cells carrying a mutated HSV-TK gene or which have lost the gene can form colonies in the presence of ACV, making it possible to detect the genetic defects in a positive manner. It is also possible to discriminate between small mutations and large deletions by checking the presence of the linked marker, neo. As a model experiment, we prepared an uncloned pool of rat fibroblast cells (CREF) infected with this virus and irradiated them with increasing doses of ultraviolet light. Dose-dependent increases in the number of ACV-resistant colonies were observed. Structural analysis of the HSV-TK gene in these clones revealed point mutations or small deletions in the majority of the cases. Since it requires no pre-existing genetic markers in the host cells, this system may be used for a wide variety of mammalian cells and provides a useful tool to assess both their susceptibility to various mutagens and their genomic instability.
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PMID:A new retroviral vector for detecting mutations and chromosomal instability in mammalian cells. 775 1


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