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:1.5.1.3 (dihydrofolate reductase)
5,819 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

We previously reported the expression of human beta-interferon (beta-IFN) (Miyaji et al., 1989) and human lymphotoxin (Miyaji et al., 1990) in Namalwa KJM-1 cells adapted to serum-free medium. To establish an efficient gene expression system, a dihydrofolate reductase (dhfr) gene coamplification method was applied to this cell line. A beta-IFN expression plasmid was introduced with a dhfr expression plasmid into KJM-1 and methotrexate (MTX)-resistant derivatives were selected by a stepwise increase of MTX concentration. Among them, derivatives which showed higher expression levels of beta-IFN than that achieved by the parental transformants were obtained, suggesting that a dhfr gene coamplification method can be used for efficient expression of foreign genes in KJM-1 which contains endogenous dhfr genes. Then, an improved beta-IFN expression vector was constructed, which contains a dhfr transcription unit. This plasmid was introduced into KJM-1 and then, MTX-resistant derivatives were selected. Among them, the highest producer, clone 40-10-24, secreted beta-IFN at a level as high as 5 micrograms/ml, which is about 100-fold higher than that obtained by the G418-resistant parental transformants. In addition, beta-IFN produced by recombinant KJM-1 cells had the same molecular weight of that produced by fibroblasts.
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PMID:Efficient expression of human beta-interferon in Namalwa KJM-1 cells adapted to serum-free medium by a dhfr gene coamplification method. 136 43

Studies were conducted to characterize the effect of gene amplification and foreign gene expression on recombinant CHO cell growth. Chinese hamster ovary (CHO) cells were transfected with an expression vector containing the gene for dihydrofolate reductase (dhfr) and the gene for human beta-interferon (beta-IFN) or the lac Z gene which codes for beta-galactosidase (beta-gal). The recombinant genes in these CHO cells were amplified stepwise by growth in 0, 10(-7), and 10(-6) M methotrexate (MTX), and the beta-gal expressing cells were adapted to suspension culture. Flow cytometric methods (FCM) were used to measure the distribution of amplified dhfr gene content and foreign beta-gal gene expression in the cell populations. A biochemical assay for beta-gal was also used. Beta-gal expression was found to increase with increasing gene amplification. The growth rate of recombinant CHO cells at 10(-7) M MTX was found to be 20% lower than that of recombinant CHO cells in MTX-free medium, and the cell growth rate at 10(-6) M MTX was 20% lower than that of recombinant CHO cells at 10(-7) M MTX. There was no effect of 10(-5) M MTX on the growth of CHO-DG44 (dhfr-) cells. The reduction of growth rate in recombinant CHO cells is therefore thought to be mainly due to the effect of dhfr and foreign gene amplification and increased beta-galactosidase expression.
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PMID:Effect of amplification of dhfr and lac Z genes on growth and beta-galactosidase expression in suspension cultures of recombinant CHO cells. 136 76

It has previously been reported that composite DNAs derived from L factor, a polyoma-related mammalian plasmid, can be established in several mouse cell lines after transfection. Here, we report that the copy number of a plasmid composite DNA consisting of L factor, pBR DNA, dihydrofolate reductase (dhfr) gene, and gamma-interferon (gamma-IFN) gene was increased more than 10-fold after two successive adaptations of the plasmid-bearing mouse L cells to increasing concentrations of methotrexate (MTX), an inhibitor of dhfr. The structure of the amplified L factor plasmid remained intact during prolonged cell culture, but the copy number remained to be amplified only when the selective pressure (presence of MTX in the medium) has been exerted during the culture. Cells bearing the amplified plasmid produced a higher level of gamma-IFN compared with the original clone, which was likely to be derived from the plasmid gamma-IFN gene amplified along with L factor and the dhfr gene.
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PMID:Amplification of plasmid (L factor) DNA and increased production of a plasmid gene product (gamma-interferon) in mouse L cells. 140 68

The gene that encodes the membrane-bound Mr 100,000 human melanoma-associated antigen (MAA) defined by mouse mAb 376.96, a leukocyte and fibroblast interferon-modulated glycoprotein having preferential distribution on melanoma and carcinoma cells, has been transfected into the mouse melanoma cell line B78H1 as a step toward molecular cloning and characterization of the MAA. Primary, secondary, and tertiary B78H1 transfectants expressing the Mr 100,000 MAA gene were generated by treatment with coprecipitated DNA from Mr 100,000 MAA+ human or transfectant mouse cells and they were detected by an indirect RBC rosetting assay. The Mr 100,000 MAA gene was also transferred into K-1735 mouse melanoma cells and into nonmalignant and malignant mouse fibroblast lines. The species immunoprecipitated by mAb 376.96 from human melanoma cells (Mr 100,000) and from mouse melanoma transfectant cells (Mr 97,000-100,000) were both converted to molecule(s) having an Mr of approximately 70,000 by enzymatic removal of asparagine-linked carbohydrate residues. Two independent secondary transformant clones of B78H1 cells express Mr 100,000 MAA antigenicity at levels significantly higher than those observed when one or two copies of the gene are present. Clone Mr 100,000 secondary-A spontaneously overexpresses Mr 100,000 MAA at least 5-fold and has greater than or equal to 10 times elevated levels of putatively Mr 100,000 MAA gene-associated human alu family repeat element (h-alu)-positive restriction fragments relative to "single" copy secondary transfectant cells. Clone Mr 100,000 secondary-B has increased copy number and expression of Mr 100,000 MAA as a consequence of a selective co-amplification procedure which is targeted to a mouse wild type dihydrofolate reductase (dhfr) gene expression vector. This vector was co-introduced into B78H1 cells in addition to the DNA of Mr 100,000 MAA+ primary transfectant cells and the initially selected aminoglycoside phosphotransferase (neo) gene vector. Stepwise selections of a secondary Mr 100,000 MAA+ transfectant clone with increasing concentrations of the dihydrofolate reductase-inhibitory antimetabolite methotrexate led to progressive increases in copy numbers of the introduced dhfr gene and to parallel increases in h-alu sequences, in cellular levels of dihydrofolate reductase protein, and in cellular mAb 376.96 reactivity. Levels of these entities ultimately reached 50-fold, relative to levels expressed prior to amplification. The array of h-alu+ restriction fragments amplified in Mr 100,000 secondary-B cell DNA is very similar to that observed in Mr 100,000 secondary-A cell DNA.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Interspecific DNA-mediated transfer and amplification of a gene specifying a Mr 100,000 human melanoma-associated cell surface glycoprotein. 230 16

In an attempt to clarify the role of beta interferon in vitro cell systems, the metabolic expression of dihydrofolate reductase (DHFR), succinate dehydrogenase (SDH) and lactate dehydrogenase (LDH), by histochemical methods was studied. DHFR was also quantified by flow cytometry. Glioblastoma cell line with or without beta interferon was used. Three days after the treatment the DHFR reaction was much less intense than in the control. Quantitative data confirmed these results. Immediately afterwards, most cells exhibited much more intense reaction. These facts underlined that, in this biological model in vitro, beta interferon could reduce the synthesis of these enzymes only for a short period.
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PMID:[Qualitative and quantitative histochemical evaluation of the dehydrogenase activity in human glioblastoma cells treated with beta-interferon]. 237 36

HindII fragment encoding human fibroblast interferon (IFN beta) gene coding sequence was fused at 60 bp downstream from the RNA start site of SV40 early gene to be a constitutive expression plasmid pSVE beta. This recombinant plasmid was transfected into the dihydrofolate reductase (dhfr)-deficient Chinese hamster ovary (CHO) cells together with a selectable dhfr gene. About half of transformants continuously secreted IFN beta into the supernatant without inducement. One of the subclone transformants constitutively produced up to 852U IFN beta/2 X 10(6) cells/ml. 48hr in common medium.
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PMID:[Constitutive expression of human fibroblast interferon gene controlled by SV40 early promoter in CHO cells]. 256 75

An expression cassette consisting of the human beta-interferon (beta-IFN) cDNA fused to the human metallothionein (MeT)IIA promoter has been linked to a selectable mouse dihydrofolate reductase gene (dhfr) and used to transform dhfr-deficient Chinese hamster ovary (CHO) cells. Transformants resistant to increasing concentrations of methotrexate (Mtx) were isolated and found to secrete beta-IFN either constitutively or upon induction with cadmium (up to 325 000 units beta-IFN/10(6) cells/24 h). Molecular analysis demonstrates a large increase in beta-IFN-specific DNA sequences and beta-IFN mRNA levels in amplified cell lines, with initiation of transcription occurring at the authentic start point for the MeT promoter.
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PMID:Expression of amplified human beta interferon genes using heavy metal induction in Chinese hamster ovary cells. 299 84

A complete cDNA clone of murine interferon-gamma (MuIFN-gamma) was obtained by recombining two appropriate segments from partial cDNA clones originally identified by colony hybridization with rat IFN-gamma chromosomal gene fragments as probes. An expression vector was constructed in which the cDNA was placed under control of the SV40 early promoter. Transient expression of MuIFN-gamma was obtained by transformation of COS-1 cells. Subsequently, this interferon expression unit was linked to a vector containing a dihydrofolate reductase (DHFR) modular gene and used to transform DHFR(-)-CHO cells. Cell clones were selected that constitutively produce an interferon activity which by several criteria was found to be indistinguishable from natural, splenocyte-derived MuIFN-gamma.
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PMID:Molecular cloning of murine interferon gamma (MuIFN-gamma) cDNA and its expression in heterologous mammalian cells. 299 40

The coding sequence of the human interferon (IFN)-beta 1 gene, fused 60 bp downstream from the RNA start site of the SV40 early gene, was transfected into dihydrofolate reductase (DHFR)-deficient Chinese hamster ovary (CHO) cells together with a selectable DHFR gene. Most transformants continuously secreted IFN-beta 1 into the medium. Induction did not stimulate expression of the fused SV40-IFN-beta 1 gene. The role of the SV40 promoter was verified by transforming cells with the unmodified human IFN-beta 1 gene, or by the IFN-beta 1 coding region fused to another poly(rI):(rC)-inducible gene. In these cases, the transformants showed strictly inducible (not constitutive) IFN secretion. By selection for methotrexate resistance, CHO clones with a 10-20-fold amplification of the SV40-IFN-beta 1 DNA were obtained. Such clones constitutively produce up to 350,000 units IFN/ml per 10(6) cells/24 hr, i.e., over 10 times more than fully induced human fibroblasts. In continuous culture with daily changes of medium, accumulation of IFN-beta 1 is constant at a rate of 300,000 molecules per cell/hr. Batches of up to 16 mg of IFN-beta 1 produced by the transformed CHO cells were purified to homogeneity by affinity chromatography on monoclonal antibodies. This IFN appears identical in size, activity, and immunospecificity to the native human IFN-beta 1 glycoprotein.
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PMID:Efficient constitutive production of human fibroblast interferon by hamster cells transformed with the IFN-beta 1 gene fused to an SV40 early promoter. 609 17

Hybrid plasmids containing the mouse dihydrofolate reductase (dhfr) and a human interferon (either IFN-alpha 5 or IFN-gamma) coding sequence under the control of viral promoters were transfected into dhfr- Chinese hamster ovary (CHO) cells. dhfr+ colonies produced IFN at 10-1000 units X ml-1 X day-1. Clones selected in methotrexate had a 20-50-fold increase in the IFN-alpha 5 and dhfr DNA and mRNA content and secreted IFN at 20,000-100,000 units X ml-1 X day-1. SDS-polyacrylamide gel electrophoresis of partially purified 35S-HuIFN-gamma from CHO cells showed a multiple of labeled bands with a mobility corresponding to 22,400 to 23,400 daltons which was absent in the supernatants of non-transformed CHO cells. The higher apparent molecular weight of human IFN-gamma from CHO cells as compared to that of human IFN-gamma from E. coli (about 18,800) suggests that the former was glycosylated.
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PMID:Constitutive, long-term production of human interferons by hamster cells containing multiple copies of a cloned interferon gene. 618 7


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