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1.
J. Schwaber 《Experimental cell research》1975,93(2):343-354
The fusion of human lymphocytes and TEPC-15 mouse myeloma cells, which had not been adapted to culture, resulted in the establishment of in vitro hybrid cell cultures. Ten clones of this somatic cell hybrid were examined. There was preferential exclusion of human chromosomes: between two and five human chromosomes were identified in the hybrid clones by Giemsa banding. All of the clones had the mouse parental histocompatibility antigens, but only four clones also retained the human parental histocompatibility antigens. Secretion of parental immunoglobulin was determined by SDS-gel electrophoresis of species-specific immune precipitates. Synthesis of parental immunoglobulin by individual hybrid cells was determined by double label fluorescent antibody staining. Individual cells from six of the clones secreted and synthesized both human and mouse parental immunoglobulins. Three clones secreted only one parental immunoglobulin. Cells from one of these clones secreted and synthesized only human immunoglobulin. Cells from the remaining two clones secreted only one parental species of immunoglobulin but synthesized both human and mouse immunoglobulins. Finally, one clone did not secrete immunoglobulin, yet the individual cells synthesized both human and mouse parental species of immunoglobulin. 相似文献
2.
Aftab Alam 《Journal of biosciences》1986,10(1):37-47
Dihydrofolate reductase has been purified from a methotrexate-resistant human lymphoid cell line (CCRF/CEM-R3) and up to 1
mg of enzyme has been obtained from 5 litres of culture. The enzyme has a molecular weight of 22000 ±500 as determined by
gel filtration. The pH activity profile shows a single optimum at pH 7.7, where marked activation is observed by addition
of 0.2 M NaCl. TheK
m for NADPH is 3μM and dihydrofolate 0.7μM. The binding constant for the inhibitor, methotrexate, is 29 pM 相似文献
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Effects of 5-fluorouracil on dihydrofolate reductase and dihydrofolate reductase mRNA from methotrexate-resistant KB cells 总被引:3,自引:0,他引:3
Growth of methotrexate-resistant dihydrofolate reductase gene-amplified KB cells in the presence of 5-fluorouracil results in an increase in dihydrofolate reductase mRNA. This increase can be solely attributed to a species of RNA of approximately 3.5 kilobase pairs in size. Although dihydrofolate reductase enzyme activity increases per cell with increasing 5-fluorouracil, there is a decrease of enzyme activity per mg of protein (Dolnick, B. J., and Pink, J. J. (1983) J. Biol. Chem. 258, 13299-13306). The rate of in vivo enzyme synthesis, as assayed by immunoprecipitation and supported by gel electrophoresis, does not decrease and may in fact increase with increasing 5-fluorouracil. Translation of purified dihydrofolate reductase mRNA in vitro shows that the rate of translation is unaffected by 5-fluorouracil incorporation into mRNA. The inhibition of dihydrofolate reductase by a monospecific polyclonal antiserum is reduced with extracts from 5-fluorouracil-treated cells. Inhibition of dihydrofolate reductase by methotrexate is significantly reduced in extracts from 5-fluorouracil-treated cells compared to control extracts. Tight binding of [3H]methotrexate is also different in extracts from 5-fluorouracil-treated cells. This data supports the hypothesis of translational miscoding during protein synthesis as a major mechanism of 5-fluorouracil-mediated cytotoxicity and suggests a new mechanism of 5-fluorouracil-methotrexate antagonism. 相似文献
5.
Increased dihydrofolate reductase activity in methotrexate-resistant human promyelocytic-leukaemia (HL-60) cells. Lack of correlation between increased activity and overproduction. 总被引:1,自引:0,他引:1
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Methotrexate(MTX)-resistant human promyelocytic-leukaemia cells (HL-60) derived from MTX-sensitive cells have a 20-fold increase in dihydrofolate reductase (DHFR) activity as compared with the sensitive cells. This increase is not associated with a concomitant increase in DHFR protein as determined by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis and by immunological methods using mouse anti-DHFR antibody. The rate of DHFR synthesis is similar in both cell lines. Furthermore, both the sensitive and resistant cells have similar amounts of RNA hybridizing to a DHFR complementary-DNA probe, correlating well with the lack of increase in DHFR protein. DHFR-gene dosages were similar in both types of cells. We conclude that the 20-fold increase in DHFR activity present in these MTX-resistant cells is not due to the overproduction of DHFR but due to the expression of a more active form of the enzyme. 相似文献
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7.
Multiple origins of replication in the dihydrofolate reductase amplicons of a methotrexate-resistant chinese hamster cell line. 总被引:9,自引:6,他引:3
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We recently showed that replication initiates in the early S period at two closely spaced zones in the 240-kilobase (kb) dihydrofolate reductase (DHFR) amplicon of the methotrexate-resistant Chinese hamster ovary cell line CHOC 400. Both of these initiation loci (ori-beta and ori-gamma) have previously been cloned in a recombinant cosmid. In this study, we identified a third early-firing initiation locus (ori-alpha) in the much larger DHFR amplicon of the independently isolated methotrexate-resistant Chinese hamster cell line DC3F-A3/4K (A3/4K). We describe the molecular cloning of this newly identified locus and demonstrate by chromosomal walking that ori-alpha lies approximately 240 kb upstream from ori-beta. Using overlapping cosmid clones for more than 450 kb of DNA sequence from this region of the DHFR domain, we have monitored the replication pattern of the amplicons in synchronized A3/4K cells. These studies suggest that ori-alpha, ori-beta, and ori-gamma are the only early-firing initiation sites in this 450-kb sequence. In addition, we have been able to roughly localize the termini between ori-alpha and ori-beta and between ori-alpha and the next origin in the 5' direction. Thus, we have now isolated the equivalent of three early-firing replicons (including their origins) from a well-characterized chromosomal domain. With these tools, it should be possible to determine those properties that are shared by the origins and termini of different replicons and which are therefore likely to be functionally significant. 相似文献
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9.
George H. Hitchings Jr. 《In vitro cellular & developmental biology. Plant》1989,25(4):303-310
The Nobel Foundation 1989. 相似文献
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12.
M Poe 《Biochemical and biophysical research communications》1973,54(3):1008-1014
Dihydrofolate reductase from strain MB 1428 of Escherichia coli was shown to catalyze the oxidative cleavage of dihydrofolate at the C(9)N(10) bond. One of the products of the reaction was identified as 7,8-dihydropterin-6-carboxaldehyde through its proton magnetic resonance spectrum. The maximal enzymatic rate was 0.05 moles dihydrofolate cleaved per minute per mole enzyme at 25° and pH 7.2, and the KM for dihydrofolate was 17.5 ± 2.5 μM. The enzymatic reaction was fully inhibitable with methotrexate. The mechanism of enzyme action was proposed to be an apparent “acidification” of dihydrofolate upon binding to the enzyme. Folate underwent an analogous oxidative cleavage by enzyme with a turnover number of 0.0014, which produced pterin-6-carboxaldehyde. Methotrexate was also slowly degraded by the enzyme. 相似文献
13.
A new selective medium has been developed for cells containing the enzyme deoxycytidine deaminase. This medium contains hypoxanthine, aminopterin, and 5-methyldeoxycytidine (HAM medium). To survive in the presence of the aminopterin, the cells must utilize deoxycytidine deaminase to convert the 5-methyldeoxycytidine to thymidine. The cells must also have thymidine kinase and hypoxanthine phosphoribosyltransferase. A mouse cell line deficient in deoxycytidine deaminase has been isolated from a deoxycytidine kinase-deficient line, using 5-bromodeoxycytidine as the selective agent. A hybrid line between this double mutant and a human diploid fibroblast was isolated in HAM medium. The hybrid line contains the chromosomes expected of a human-mouse hybrid. The deoxycytidine deaminase isozyme patterns on cellogel show that the human-mouse hybrid cell line produces an enzyme with an electrophoretic mobility intermediate between that of the human and that of the mouse. 相似文献
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15.
Fossati E Volpato JP Poulin L Guerrero V Dugas DA Pelletier JN 《Journal of biomolecular screening》2008,13(6):504-514
We report a rapid and reliable 2-tier selection and screen for detection of activity as well as drug-resistance in mutated variants of a clinically-relevant drug-target enzyme. Human dihydrofolate reductase point-mutant libraries were subjected to a 1st-tier bacterial complementation assay, such that bacterial propagation served as an indicator of enzyme activity. Alternatively, when selection was performed in the presence of the inhibitor methotrexate (MTX), propagation indicated MTX resistance. The selected variants were then subjected to a 2nd-tier in vitro screen in 96-well plate format using crude bacterial lysate. Conditions were defined to establish a threshold for activity or for MTX resistance. The 2nd-tier assay allowed rapid detection of the best variants among the leads and provided reliable estimates of relative reactivity, (k(cat)) and IC(50)(MTX). Screening saturation libraries of active-site positions 7, 15, 24, 70, and 115 revealed a variety of novel mutations compatible with reactivity as well as 2 novel MTX-resistant variants: V115A and V115C. Both variants displayed K(i)(MTX)=20 nM, a 600-fold increase relative to the wild-type. We also present preliminary results from screening against further antifolates following simple modifications of the protocol. The flexibility and robustness of this method will provide new insights into interactions between ligands and active-site residues of this clinically relevant human enzyme. 相似文献
16.
Nucleotide sequence of the dihydrofolate reductase gene of methotrexate-resistant Lactobacillus casei 总被引:4,自引:0,他引:4
J Andrews G M Clore R W Davies A M Gronenborn B Gronenborn D Kalderon P C Papadopoulos S Sch?fer P F Sims R Stancombe 《Gene》1985,35(1-2):217-222
The nucleotide sequence of the dihydrofolate reductase (DHFR) gene of a methotrexate-resistant strain of Lactobacillus casei, which is the source of DHFR for nuclear magnetic resonance (NMR) studies, has been determined. The derived amino acid sequence differs from that obtained by protein sequencing by the presence of aspartic acid instead of asparagine at position 8 and proline instead of leucine at position 90. The nucleotide sequences of 320-bp 5' and 335-bp 3' flanking regions of this gene have also been determined. 相似文献
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18.
5-fluorouracil modulation of dihydrofolate reductase RNA levels in methotrexate-resistant KB cells 总被引:5,自引:0,他引:5
Cytotoxicity and growth inhibition by 5-fluorouracil in methotrexate-resistant dihydrofolate reductase gene-amplified KB cells in the presence of 30 microM thymidine correlates with incorporation of this fluorinated pyrimidine into RNA. Growth of these cells over several generations in the presence of inhibitory concentrations of 5-fluorouracil does not depress the steady state levels of either 18 or 28 S RNA but actually causes an increase. Similarly the rates of RNA and protein synthesis in 5-fluorouracil-treated cells are not decreased. The level of dihydrofolate reductase RNA from 5-fluorouracil-treated cells increases in a dose-dependent manner correlated with 5-fluorouracil incorporation into RNA. The qualitative size distribution of the dihydrofolate reductase RNA species is unaffected when examined by the Northern blotting technique indicating an RNA processing lesion is not induced by 5-fluorouracil incorporation into RNA. As the dose of dihydrofolate reductase RNA increases, there is no change in the level of dihydrofolate reductase specific activity, but the level of enzyme activity per cell increases. The relevance of these phenomena to the mechanism of 5-fluorouracil effect on RNA and relevance to combination chemotherapy with methotrexate are discussed. 相似文献
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20.
Organization and genesis of dihydrofolate reductase amplicons in the genome of a methotrexate-resistant Chinese hamster ovary cell line. 总被引:22,自引:14,他引:8
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We have recently isolated overlapping recombinant cosmids that represent the equivalent of two complete dihydrofolate reductase (dhfr) amplicon types from the methotrexate-resistant Chinese hamster ovary (CHO) cell line CHOC 400. In the work described in this report, we used pulse-field gradient gel electrophoresis to analyze large SfiI restriction fragments arising from the amplified dhfr domains. The junction between the 260-kilobase type I amplicons (which are arranged in head-to-tail configurations in the genome) has been localized, allowing the construction of a linear map of the parental dhfr locus. We also show that the 220-kilobase type II amplicons are arranged as inverted repeat structures in the CHOC 400 genome and arose from the type I sequence relatively early in the amplification process. Our data indicate that there are a number of minor amplicon types in the CHOC 400 cell line that were not detected in previous studies; however, the type II amplicons represent ca. 75% of all the amplicons in the CHOC 400 genome. Both the type I and type II amplicons are shown to be composed entirely of sequences that were present in the parental dhfr locus. Studies of less resistant cell lines show that initial amplicons can be larger than those observed in CHOC 400. Once established, a given amplicon type appears to be relatively stable throughout subsequent amplification steps. We also present a modification of an in-gel renaturation method that gives a relatively complete picture of the size and variability of amplicons in the genome. 相似文献