Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:1.5.1.3 (dihydrofolate reductase)
 5,819 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Expression of certain variants of dihydrofolate reductase (DHFR) in mammalian cells protects them from methotrexate. Retroviral transfer of the gene for such a variant DHFR into hematopoietic cells might permit selection of modified cells in vivo by antifolate administration or alleviate antifolate-induced myelosuppression in patients receiving antifolate therapy. We examined protection of cells of the human lymphoblastoid line, CCRF-CEM, transduced with variants of mouse DHFR. In transduced cells selected with G418 but not with antifolate, the variant that had arginine substituted for leucine 22 did not protect against either methotrexate or trimetrexate; however, four other variants did offer protection, with the best having leucine 22 changed to tyrosine. Polyclonal cultures transduced with the different variants express DHFR at about the same level, but clones within each polyclonal population differ in DHFR expression levels and in resistance. These differences in expression were shown to reflect different integration sites for proviral DNA. Exposure to trimetrexate selects highly resistant clones, with high expression due to both high copy number and integration sites that are favorable for expression. Differences in the resistance of cultures expressing different variants at the same level are due to differences in the catalytic activity of the expressed DHFR, its affinity for antifolates, and its stability.
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PMID:Protection of CCRF-CEM human lymphoid cells from antifolates by retroviral gene transfer of variants of murine dihydrofolate reductase. 969 74

We investigated whether transduction of human cord blood progenitor cells can be increased by spinoculation in fibronectin fragment CH-296 (FN)-coated tubes. Bicistronic vectors PA317/LgEIN, containing the enhanced green fluorescent protein (EGFP) and neomycin phosphotransferase (neo) genes, and PG13/LgDIN, containing the dihydrofolate reductase and neo genes, were used to transduce CD34-enriched human cord blood cells. Transduction by spinoculation in FN-coated tubes (spin/FN+) was compared with spinoculation in noncoated tubes (spin/FN-) and transduction in plates coated with FN (plate/FN+). Antibody to TGF-beta was added to spin/FN+ to evaluate its impact on transduction. Using producer cell line PA317/LgEIN for transduction of CD34+ cord blood cells, FACS analysis for expression of EGFP revealed mean transduction of 30.6+/-4.3, 9.1+/-1.6, and 21.1+/-6.5% of CD34+ cells in the spin/FN+, spin/FN-, and plate/FN+ arms, respectively. Transduction of CD+CD38low cells was also higher in the spin/FN+ arm as compared with transduction in the spin/FN- arm. These results were corroborated by colony-forming assays. Antibody to TGF-beta did not further increase transduction. Using a different producer cell line, PG13/pLgDIN, a higher number of G418-resistant CFU-GM was observed in the spin/FN+ as compared with the plate/FN+ and spin/FN-arms. NOD/SCID mice were transplanted with transduced, CD34-enriched human cord blood cells, and persistence of transduced human cells was analyzed in the mice marrows after 6-8 weeks: 32.8, 6.0, and 23.9% human G418-resistant CFU-GM colonies were observed in the spin/FN+, spin/FN-, and plate/FN+ arms, respectively. These results suggest that spinoculation in FN-coated tubes increases transduction of early human cord blood progenitor cells as compared with spinoculation in noncoated tubes.
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PMID:Increased gene transfer into human cord blood cells by centrifugation-enhanced transduction in fibronectin fragment-coated tubes. 1058 31

pYACneo, a 15.8-kb plasmid, contains a bacterial origin, G418-resistance gene, and yeast ARS, CEN, and TEL elements. Three mammalian origins have been cloned into this circular vector: 343, a 448-bp chromosomal origin from a transcribed region of human chromosome 6q; X24, a 4.3-kb element containing the hamster DHFR origin of bidirectional replication (oribeta), and S3, a 1.1-kb human anti-cruciform purified autonomously replicating sequence. The resulting constructs have been transfected into HeLa cells, and G418-resistant subcultures were isolated. The frequency of G418-resistant transformation was 1.7-8.7 times higher with origin-containing YACneo than with vector alone. After >45 generations under G418 selection, the presence of episomal versus integrated constructs was assessed by fluctuation assay and by PCR of supercoiled, circular, and linear genomic cellular DNAs separated on ethidium bromide-cesium chloride gradients. In stable G418-resistant subcultures transfected with vector alone or with linearized constructs, as well as in some subcultures transfected with circular origin-containing constructs, resistance was conferred by integration into the host genome. However, several examples were found of G418-resistant transfectants maintaining the Y.343 and the YAC.S3 circular constructs in a strictly episomal state after long-term culture in selective medium, with 80-90% stability per cell division. The episomes were found to replicate semiconservatively in a bromodeoxyuridine pulse-labeling assay for </=130 cell generations after transfection. Furthermore, after </=172 cell generations rescued episomal DNA could be isolated intact and unrearranged, and could be used to retransform bacteria. These versatile constructs, containing mammalian origins, have the capacity for further modification with human telomere or large putative centromere elements, in an effort to move towards construction of a human artificial chromosome.
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PMID:Circular YAC vectors containing short mammalian origin sequences are maintained under selection as HeLa episomes. 1065 86

Sleeping Beauty (SB) is a gene-insertion system reconstructed from transposon sequences found in teleost fish and is capable of mediating the transposition of DNA sequences from transfected plasmids into the chromosomes of vertebrate cell populations. The SB system consists of a transposon, made up of a gene of interest flanked by transposon inverted repeats, and a source of transposase. Here we carried out a series of studies to further characterize SB-mediated transposition as a tool for gene transfer to chromosomes and ultimately for human gene therapy. Transfection of mouse 3T3 cells, HeLa cells, and human A549 lung carcinoma cells with a transposon containing the neomycin phosphotransferase (NEO) gene resulted in a several-fold increase in drug-resistant colony formation when co-transfected with a plasmid expressing the SB transposase. A transposon containing a methotrexate-resistant dihydrofolate reductase gene was also found to confer an increased frequency of methotrexate-resistant colony formation when co-transfected with SB transposase-encoding plasmid. A plasmid containing a herpes simplex virus thymidine kinase gene as well as a transposon containing a NEO gene was used for counterselection against random recombinants (NEO+TK+) in medium containing G418 plus ganciclovir. Effective counterselection required a recovery period of 5 days after transfection before shifting into medium containing ganciclovir to allow time for transiently expressed thymidine kinase activity to subside in cells not stably transfected. Southern analysis of clonal isolates indicated a shift from random recombination events toward transposition events when clones were isolated in medium containing ganciclovir as well as G418. We found that including both transposon and transposase functions on the same plasmid substantially increased the stable gene transfer frequency in Huh7 human hepatoma cells. The results from these experiments contribute technical and conceptual insight into the process of transposition in mammalian cells, and into the optimal provision of transposon and transposase functions that may be applicable to gene therapy studies.
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PMID:Counterselection and co-delivery of transposon and transposase functions for Sleeping Beauty-mediated transposition in cultured mammalian cells. 1615 96

Opine synthases are enzymes produced in dicotyledonous plants as the result of a natural gene transfer phenomenon. Agrobacteria contain Ti plasmids that direct the transfer, stable integration and expression of a number of genes in plants, including the genes coding for octopine or nopaline synthase. This fact was used as the basis for the construction of a number of chimeric genes combining the 5' upstream promoter sequences and most of the untranslated leader sequence of the nopaline synthase (nos) gene with the coding sequence of two bacterial genes: the aminoglycoside phosphotransferase (APH(3')II) gene of Tn5 and the methotrexate-insensitive dihydrofolate reductase (DHFR Mtx) of the R67 plasmid. The APH(3')II enzyme inactivates a number of aminoglycoside antibiotics such as kanamycin, neomycin and G418. Kanamycin, G418 and methotrexate are very toxic to plants. The chimeric NOS-APH(3')II gene, when transferred to tobacco cells using the Ti plasmid as a gene vector, was expressed and conferred resistance to kanamycin to the plant cells. Kanamycin-resistant tobacco cells were shown to contain a typical APH(3')II phosphorylase activity. This chimeric gene can be used as a potent dominant selectable marker in plants. Similar results were also obtained with a NOS-DHFR Mtx gene. Our results demonstrate that foreign genes are not only transferred but are also functionally expressed when the appropriate constructions are made using promoters known to be active in plant cells.
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PMID:Chimeric genes as dominant selectable markers in plant cells. 1645 64


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