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)

Repetitive DNA sequences have been implicated in the mediation of DNA rearrangement in mammalian cells. We have tested this hypothesis by using a dihydrofolate reductase (DHFR) expression vector into which candidate sequences were inserted. DHFR- Chinese hamster ovary (CHO) cells were transfected with this vector, the amplification of which was then selected for by methotrexate (MTX) exposure. Cells transfected with the vector alone (and resistant to 0.02 or 1.0 microM MTX) or with a poly(dG-dT) insert (and resistant to 0.05 or 1.0 microM MTX) showed little change in chromosome aberrations or sister chromatid exchange frequencies. In contrast, transfection of DHFR- CHO cells with a vector containing either of two distinct 0.34-kilobase human alphoid DNA segments (and selection to 0.05 to 10.0 microM MTX) showed an approximately 50% increase in chromosome number and marked changes in chromosome structure, including one or two dicentric or ring forms per cell. The sister chromatid exchange frequency also increased, to more than double the frequency of that in cells transfected without insert or those containing poly(dG-dT). In situ hybridization of one 0.34-kilobase insert in some cells suggested clustering of homologous sequences in structurally abnormal recipient CHO cell chromosomes. The approach described provides an introduction to a unique means for a coordinate molecular and cytological study of dynamic changes in chromosome structure.
Mol Cell Biol 1988 Sep
PMID:Chromosome instability associated with human alphoid DNA transfected into the Chinese hamster genome. 322 60

A method for the measurement of methotrexate based on its inhibition of dihydrofolate reductase (EC 1.5.1.3) is described. The performance of the method is evaluated and a brief assessment of its clinical usefulness made. Reaction time, pH, and the relative concentration of enzyme and inhibitor all have a significant effect on the shape of the standard curve. Pre-incubation of enzyme with methotrexate resulted in increased inhibition, but did not improve the sensitivity or linearity of the assay. Data are presented on the imprecision, specificity and accuracy of the method. The method is simple to perform, rapid and inexpensive. The sensitivity and specificity of the method are such as to allow for effective monitoring of patients on methotrexate therapy.
Ann Clin Biochem 1988 Sep
PMID:Development and evaluation of an enzyme inhibition assay for methotrexate. 323 54

The human 4F2 cell surface antigen is a 120-kilodalton (kDa) disulfide-linked heterodimer which is composed of an 80- to 90-kDa glycosylated heavy chain (4F2HC) and a 35- to 40-kDa nonglycosylated light chain (4F2LC). 4F2 belongs to a family of inducible cell surface molecules which are involved in T-lymphocyte activation and growth. To better understand the molecular mechanism(s) that controls 4F2HC gene expression in both resting and activated T cells, a 4F2HC human genomic clone was isolated and structurally characterized. The 4F2HC gene spans 8 kilobases of chromosome 11 and is composed of nine exons. The 5' upstream region of the gene displays several properties which are characteristic of housekeeping genes. It is G+C rich and hypomethylated in peripheral blood lymphocyte DNA and contains multiple binding sites for the Sp1 transcription factor while lacking TATA or CCAAT sequences. This region of the gene also displays sequence homologies with several other inducible T-cell genes, including the interleukin-2, interleukin-2 receptor alpha chain, dihydrofolate reductase, thymidine kinase, and transferrin receptor genes. A 255-base-pair fragment of the 4F2HC gene which contains 154 base pairs of the 5' flanking sequence was able to efficiently promote expression of the bacterial chloramphenicol acetyltransferase gene in human Jurkat T cells, indicating that it contains promoter or enhancer (or both) sequences. Analyses of chromatin structure in resting and lectin-activated T cells revealed the presence of stable DNase I-hypersensitive sites within both the 5' flanking and intron 1 regions of the 4F2HC gene. Although the 4F2HC gene displayed many of the structural features characteristic of a constitutively expressed gene, lectin-mediated activation of resting peripheral blood T lymphocytes resulted in a dramatic increase in steady-state levels of 4F2HC mRNA.
Mol Cell Biol 1988 Sep
PMID:Isolation and structural characterization of the human 4F2 heavy-chain gene, an inducible gene involved in T-lymphocyte activation. 326 70

The secondary and tertiary structure of T4 bacteriophage dihydrofolate reductase is investigated by vacuum ultraviolet circular dichroism (CD) spectroscopy and probability analysis of the primary amino acid sequence. The far ultraviolet CD spectrum of the enzyme in the range of 260-178 nm is analyzed by the generalized inverse and variable selection methods developed by our laboratory. Variable selection yields an average content of 26% alpha-helix, 21% antiparallel beta-sheet, 10% parallel beta-sheet, 20% beta-turns, and 32% "other" structures within the T4 protein. The characteristic peaks of the CD spectrum indicate that the enzyme has a lot of antiparallel beta-sheet, which is typical of the alpha + beta tertiary class of globular proteins. The secondary structure of the protein is also analyzed by using four statistical methods on the amino acid sequence. Although the secondary structures predicted by each individual statistical method vary to a considerable extent, the fractions of each structure jointly predicted by a majority of the methods are in excellent agreement with our CD analysis. The alternating arrangement for some segments of alpha-helix and beta-sheet predicted from primary structure to be within the enzyme is characteristic of proteins containing parallel beta-sheet. This supports our conclusion that the protein contains both parallel and antiparallel beta-sheet structures, but finding both types of beta-sheet also means that the protein may have the variation on alpha/beta tertiary structure recently found in EcoRI endonuclease and thymidylate synthase. These observations, in conjunction with other physical properties of the T4 reductase, suggest that the enzyme perhaps shares an evolution in common with the dihydrofolate reductases derived from type I R-plasmids rather than with the host-cell protein.
J Biol Chem 1987 Sep 25
PMID:The conformation of T4 bacteriophage dihydrofolate reductase from circular dichroism. 330 67

The chiral complex tris (diphenylphenanthroline) cobalt (III) (Co(DIP)3(3+) provides a photoreactive probe for chromatin structure in mammalian cells. The complex, which upon photoactivation cleaves DNA in a conformation-specific fashion in vitro, is shown also to cleave DNA in vivo upon irradiation with ultraviolet light (greater than 300 nm). delta- and lambda-Co (DIP)3(3+) isomers are taken up efficiently into cultured Chinese hamster ovary cells and concentrate within cell nuclei. In the absence of light the complexes are toxic to the cells (10% survival at approximately 300 nM), but after ultraviolet irradiation, the toxicity is markedly (greater than 10-fold) increased. The synergism between irradiation and Co(DIP)3(3+) administration may lie in photoreactions with DNA elicited by the cobalt complex. Alkaline sucrose gradient analysis of DNA from cells exposed to lambda-Co(DIP)3(3+) and irradiation show single-stranded DNA fragmentation under conditions where little cleavage is seen in cells either incubated with lambda-Co(DIP)3(3+) or irradiated with greater than 300 nm A ultraviolet light. Cellular DNA is cleaved with lower efficiency than naked DNA, likely due to decreased accessibility of sites in vivo. Hybridization of fragments obtained from the alkaline sucrose gradients to a probe specific for the amplified dihydrofolate reductase gene reveals a similar distribution of dhfr sequences and total DNA, indicating that the family of conformations recognized by lambda-Co(DIP)3(3+) are dispersed throughout the genome.
Mutat Res 1988 Sep
PMID:In vivo photoreaction of a chiral cobalt complex: DNA cleavage by Co(DIP)3(3+) in mammalian cells. 341 45

A flow microcalorimetric method was developed for the analysis of enzymatic activities in crude tissue homogenates. It can be applied whenever a heat exchange is involved in an enzymatic reaction. The consequent sensitivity obviously depends on the enthalpy variation observed. Dihydrofolate reductase was chosen as an example; this enzyme is the molecular target of methotrexate, a widely used anticancer agent. This calorimetric method, whose sensitivity limit is 1.48 X 10(-4) units of dihydrofolate reductase per milliliter of reactant medium, allows enzyme activity measurements in tissues with low dihydrofolate reductase levels. A few examples of measurements in animal tissues are given. These measurements are of some interest; indeed, increased activity and increased levels of this enzyme are two of the mechanisms which may explain resistance to methotrexate.
Anal Biochem 1987 Sep
PMID:A flow microcalorimetric method for enzyme activity measurements: application to dihydrofolate reductase. 342 3

Lipophilic gamma-monoamide derivatives of aminopterin (AMT) were synthesized in high overall yield from 4-amino-4-deoxy-N10-formylpteroic acid and gamma-N-tert-alkyl-, gamma-N-aralkyl-, or gamma-N-arylamides of alpha-benzyl L-glutamate via a modification of the mixed carboxylic-carbonic anhydride coupling method. Coupling was also accomplished with p-nitrophenyl 4-amino-4-deoxy-N10-formylpteroate. Compounds obtained in this manner included the gamma-tert-butylamide, gamma-(1-adamantylamide), gamma-benzylamide, gamma-(3,4-dichlorobenzylamide), gamma-(2,6-dichlorobenzylamide), gamma-anilide, gamma-(3,4-methylenedioxyanilide), and gamma-(3,4-dihydroxanilide) derivatives of AMT. Also prepared, from 4-amino-4-deoxy-N10-methylpteroic acid via diethyl phosphorocyanidate coupling, was the gamma-(3,4-methylenedioxyanilide) of MTX. The methylenedioxyanilides were cleaved smoothly to dihydroxyanilides with boron tris(trifluoroacetate) in trifluoroacetic acid. All the gamma-monoamides were tested as inhibitors of purified dihydrofolate reductase (DHFR) from murine L1210 leukemia cells and as inhibitors of the growth of wild-type L1210 cells and a subline (L1210/R81) with high-level resistance to MTX and AMT based mainly on a defect in drug uptake via active transport. Several compounds were also tested against human leukemic lymphoblasts (CEM cells) and a resistant subline (CEM/MTX) whose resistance is likewise based on uptake. The IC50 of the gamma-monoamides against DHFR was 1.5- to 5-fold higher than that of the parent acids, but the IC50 against cultured cells varied over a much broader range, suggesting that uptake and/or metabolism rather than DHFR binding are principal determinants of in vitro growth inhibitory activity for these compounds. gamma-N-Aryl and gamma-N-aralkyl derivatives appeared to be more potent than gamma-N-tert-alkyl derivatives. Where comparison could be made, AMT gamma-monoamides were more potent than MTX gamma-monoamides. Several of the gamma-monoamides showed potency comparable to that of the parent acid against wild-type L1210 and CEM cells; all of them were more potent than MTX against the L1210/R81 subline; and some of the AMT gamma-monoamides were also more potent than the parent acid against resistant CEM/MTX cells. As a group, however, the gamma-monoamides were considerably more active against the murine cells than against the human cells, suggesting that the former may take up the amides better or may be able to metabolize them more efficiently than the parent acids. All the gamma-monoamides were tested in vivo against L1210 leukemia in mice.(ABSTRACT TRUNCATED AT 400 WORDS)
J Med Chem 1986 Sep
PMID:Methotrexate analogues. 28. Synthesis and biological evaluation of new gamma-monoamides of aminopterin and methotrexate. 346 94

The major alternative polyadenylation sites in the Chinese hamster dihydrofolate reductase (dhfr) gene have been identified by DNA sequencing and RNase protection experiments. Comparison of the 3' gene sequence and polyadenylation sites with those of the mouse reveals that, despite an overall sequence homology, the major sites are different in the two species. A series of minigenes was constructed containing the dhfr promoter and the first intron but lacking the four large introns of the genomic sequence. These minigenes contained either all three polyadenylation sites, no polyadenylation sites, or just the first site. All of these minigenes, as well as a cosmid clone containing the full genomic sequence, could transform DHFR-deficient Chinese hamster ovary cell mutants to a DHFR-positive phenotype with approximately equal efficiencies. A minigene lacking the first intron was markedly less efficient. Analysis of dhfr mRNA from transfectant clones derived from minigenes showed that the dhfr polyadenylation sites were used when included, but novel sites were often used in addition. When endogenous polyadenylation sites were absent, new sites in flanking carrier or host DNA were recruited. Transfectants produced by the full genomic dhfr gene yielded mRNA species that were identical in size and relative abundance to the endogenous dhfr gene. The results indicate that the minimal signals for polyadenylation are not complex and can be easily acquired from foreign sequences.
Somat Cell Mol Genet 1987 Sep
PMID:Polyadenylation of Chinese hamster dihydrofolate reductase genomic genes and minigenes after gene transfer. 347 73

The urea-induced equilibrium unfolding transition of dihydrofolate reductase from Escherichia coli was monitored by UV difference, circular dichroism (CD), and fluorescence spectroscopy. Each of these data sets were well described by a two-state unfolding model involving only native and unfolded forms. The free energy of folding in the absence of urea at pH 7.8, 15 degrees C is 6.13 +/- 0.36 kcal mol-1 by difference UV, 5.32 +/- 0.67 kcal mol-1 by CD, and 5.42 +/- 1.04 kcal mol-1 by fluorescence spectroscopy. The midpoints for the difference UV, CD, and fluorescence transitions are 3.12, 3.08, and 3.18 M urea, respectively. The near-coincidence of the unfolding transitions monitored by these three techniques also supports the assignment of a two-state model for the equilibrium results. Kinetic studies of the unfolding and refolding reactions show that the process is complex and therefore that additional species must be present. Unfolding jumps in the absence of potassium chloride revealed two slow phases which account for all of the amplitude predicted by equilibrium experiments. Unfolding in the presence of 400 mM KCl results in the selective loss of the slower phase, implying that there are two native forms present in equilibrium prior to unfolding. Five reactions were observed in refolding: two slow phases designated tau 1 and tau 2 that correspond to the slow phases in unfolding and three faster reactions designated tau 3, tau 4, and tau 5 that were followed by stopped-flow techniques. The kinetics of the recovery of the native form was monitored by following the binding of methotrexate, a tight-binding inhibitor of dihydrofolate reductase, at 380 nm.(ABSTRACT TRUNCATED AT 250 WORDS)
Biochemistry 1986 Sep 23
PMID:Folding of dihydrofolate reductase from Escherichia coli. 353 77

We have previously demonstrated that the active dihydrofolate reductase (DHFR) gene is efficiently repaired in Chinese hamster ovary (CHO) cells which remove only a small fraction of u.v.-induced pyrimidine dimers from the overall genome. Preferential DNA repair of essential genes may explain why the u.v. resistance of normal CHO cells is as high as that of fully repair-proficient normal human cells. In this report, we have studied the removal of pyrimidine dimers in a CHO cell line expressing the cloned denV gene from bacteriophage T4 which codes for the pyrimidine dimer specific enzyme T4 endonuclease V (T4 endo V). This cell line was derived from a u.v.-sensitive excision deficient mutant of a CHO wild type line by transformation with the denV gene, and partial restoration of u.v. resistance was achieved. We have examined an important aspect of the u.v. excision repair in these denV+ cells by studying the repair efficiencies in the active DHFR gene and in a non-coding sequence located downstream from it. In the u.v.-sensitive CHO mutant cell line from which the denV+ was derived, we detected no pyrimidine dimer removal from the gene or from the downstream sequence after irradiation of the cells with 20 J/m2 u.v. (254 nm) light. In the wild type CHO cells, approximately 50% of the pyrimidine dimers were removed from a sequence in the DHFR gene within 8 h, whereas none were removed from the downstream sequence in that period. This represents the normal pattern of preferential DNA repair of active genes, which we have described in previous communications. In the denV+ cells, approximately 70% of the pyrimidine dimers were removed from both the DHFR gene and from the downstream sequence; these cells thus repair both coding and non-coding regions of the genome and show no pattern of preferential repair. The endogenous activity that initiates excision repair in normal CHO cells is evidently much more restricted in its accessibility to DNA lesions in chromatin than is the activity in cells containing substantial amounts of the small T4 endo V enzyme.
Carcinogenesis 1987 Sep
PMID:Enhanced repair of pyrimidine dimers in coding and non-coding genomic sequences in CHO cells expressing a prokaryotic DNA repair gene. 362 70


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