Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UNIPROT:P06889 (Mol)
 630,302 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Three spontaneous fol regulatory mutants contain dihydrofolate reductase molecules which differ in physical properties from enzymes of their parent strains. The enzymes were purified over 100-fold by affinity chromatography and were shown to differ in vitro in thermolability and in affinity for trimethoprim, a competitive inhibitor of the enzyme. These results indicate that some fol regulatroy mutations occur in the structural gene for dihydrofolate reductase.
Mol Gen Genet 1977 Mar 07
PMID:Altered dihydrofolate reductase in fol regulatroy mutants of Escherichia coli K12. 32 67

DNA fragments of the R factor R388 which renders E. coli resistant to trimethoprim by inducing a trimethoprim resistant dihydrofolate reductase (Amyes and Smith, 1974) were inserted into plasmids and screened for the expression of the trimethoprim resistance gene. By means of a two step deletion procedure a 1770 bp EcoRI/BamH1 fragment was isolated which conferred drug resistance and which was found to induce the synthesis of the same dihydrofolate reductase as the parental R factor. Gene dosage experiments indicated that the induction was due to the presence of a dihydrofolate reductase structural gene on the 1770 bp fragment. The gene could be assigned to a segment which was less than 1200 bp long. The 1770 bp fragment and a recombinant plasmid consisting of pSF2124 and part of R388 were mapped with several restriction nucleases. The R factor induced enzyme was partially purified from a strain carrying a multicopy recombinant plasmid into which the 1770 bp fragment was inserted and which induced high levels of dihydrofolate reductase. The enzyme was found to be stable at 100 degrees. Some aspects of the synthesis of dihydrofolate reductase are discussed.
Mol Gen Genet 1978 Aug 04
PMID:Isolation of a small DNA fragment carrying the gene for a dihydrofolate reductase from a trimethoprim resistance factor. 36 38

Two clones from the Clarke-Carbon Escherichia coli colony bank were resistant to inhibition by trimethoprim, a potent inhibitor of dihydrofolate reductase. Both clones had elevated levels of dihydrofolate reductase. Furthermore, trimethoprim resistance and elevated enzyme levels were associated with ColE1 plasmids that carried DNA from the trkC ksgA pdxA region of the E. coli chromosome. Plasmid pLC1437a was shown by two criteria to carry the structural gene for dihydrofolate reductase: 1) A partial diploid containing plasmid pLC1437a produced a kinetically-recognizable dihydrofolate reductase that was not present in the parent haploid strain. 2) Plasmid pLC1437a coded for dihydrofolate reductase in vitro. A 1,000 base pair fragment of plasmid pLC1437a containing fol was used as a probe to measure fol mRNA in a mutant strain isolated by Sheldon and Brenner (Molec. gen. Genet. 147, 91-97, 1976). The mutation in this strain, which results in constitutively-high levels of dihydrofolate reductase and in the inability of the strain to grow at 42 degrees C, is cis dominant (Sheldon and Brenner, 1976). The results of kinetic hybridization and pulse-labeling experiments indicated that the regulatory mutant produced elevated levels of dihydrofolate reductase in response to an increased rate of synthesis of fol mRNA.
Mol Gen Genet 1979 Aug
PMID:Regulation of dihydrofolate reductase synthesis in Escherichia coli. 39 Mar 4

Trimethoprim inhibits dihydrofolate reductase. Mutations conferring trimethorpim-resistance on E coli K12 result in either an altered reductase with decreased affinity for the drug, or in 2-30 fold higher levels of the enzyme. Studies of the latter class of mutants indicate that dihydrofolate reductase is regulatdd by a diffusible molecule, and is probably under negative control. The regulatrory mutants, some of which are temperature-sensitive, act cis.
Mol Gen Genet 1976 Aug 10
PMID:Regulatory mutants of dihydrofolate reductase in Escherichia coli K12. 78 30

Recent advances in a new method for the de novo design of enzyme inhibitors are reported. A new set of rules to define the possible nonbonded contacts between protein and ligand is presented. This method was derived from published statistical analyses of nonbonded contacts in crystal packings of organic molecules and has been implemented in the recently described computer program LUDI. Moreover, LUDI can now append a new substituent onto an already existing ligand. Applications are reported for the design of inhibitors of HIV protease and dihydrofolate reductase. The results demonstrate that LUDI is indeed capable of designing new ligands with improved binding when compared to the reference compound.
J Comput Aided Mol Des 1992 Dec
PMID:LUDI: rule-based automatic design of new substituents for enzyme inhibitor leads. 129 28

The adenovirus immediate-early protein E1A activates the adenovirus E2 promoter and several cellular gene promoters through transcription factor E2F. The immediate-early proteins of human cytomegalovirus (HCMV) can complement an E1A-deficient adenovirus mutant and activate the adenovirus E2 promoter. HCMV also has been shown to activate the adenovirus E2 promoter. On the basis of these findings, we have investigated whether HCMV can activate the promoter of the cellular dihydrofolate reductase (DHFR) gene, which requires E2F binding for maximal promoter activity. We show that HCMV activates the DHFR promoter and that products of the HCMV major immediate-early gene region mediate the activation of the promoter specifically through the E2F site. We used gel mobility shift assays to search for potential molecular mechanisms for this activation and found an "infection-specific" multimeric complex that bound to the E2F sites in the DHFR and E2 promoters in extracts from HCMV-infected cells but not in extracts from uninfected cells. Several antibodies against HCMV immediate-early gene products had no effect on this infection-specific complex. Subsequently, the complex was found to contain E2F, cyclin A, p33cdk2, and p107 and to be similar to S-phase-specific complexes that recently have been identified in several cell types. A functional role for the binding of the cyclin A-p33cdk2 complex to cellular gene promoters has yet to be demonstrated; however, HCMV infection causes the induction of both cellular DNA replication and transcription of growth-related genes containing E2F sites in their promoters. The findings described above therefore may relate to both of these effects of HCMV infection. We also provide evidence that some of the molecular events associated with adenovirus infection are different from those associated with HCMV infection.
Mol Cell Biol 1992 Oct
PMID:E2F mediates dihydrofolate reductase promoter activation and multiprotein complex formation in human cytomegalovirus infection. 132 53

Gene amplification contributes to carcinogenesis by enhancing proto-oncogene activity and causing chromosomal instability. The ease of detecting amplified tumor-virus sequences has encouraged use of this system as a surrogate for studying the molecular events involved in endogenous gene amplification. We report here a new system for studying carcinogen-induced amplification of both endogenous and viral sequences in the SV40-transformed human keratinocyte line AG06. Treatment with carcinogens induced a transient dose-dependent amplification of the integrated SV40 sequences. The amplified sequences appeared in the extrachromosomal fraction. Treatment of these cells with carcinogens prior to methotrexate (MTX) selection increased the frequency of MTX-resistant colonies, 67% of which exhibited dihydrofolate reductase (dhfr) amplification. The abilities of five carcinogens with different DNA-damaging activities (the DNA-damaging agents N-methyl-N-nitro-N-nitrosoguanidine, mitomycin C (MMC), ultraviolet light C, and X-rays and the non-DNA-damaging agent arsenite) to induce SV40 and dhfr amplification at concentrations that result in 50% clonal survival were compared. All four DNA-damaging carcinogens (as well as growth arrest) were able to elicit some SV40 amplification, but responses varied markedly, from 1.8-fold for X-rays to sevenfold to eightfold for MMC. There was no correlation between the ability to elicit the two amplification responses. Arsenite, which did not induce SV40 amplification, was the best inducer of MTX resistance. These results point to different controls involved in the induction of viral and dhfr amplification. The signal for amplification of viral genes may be triggered by DNA damage and growth arrest, whereas amplification of dhfr, and perhaps other endogenous sequences, seems to be triggered by other signals as well.
Mol Carcinog 1992
PMID:Differential susceptibility to carcinogen-induced amplification of SV40 and dhfr sequences in SV40-transformed human keratinocytes. 133 30

We have studied several aspects of DNA damage formation and repair in human ovarian cancer cell lines which have become resistant to cisplatin through continued exposure to the anticancer drug. The resistant cell lines A2780/cp70 and 2008/c13*5.25 were compared with their respective parental cell lines, A2780 and 2008. Cells in culture were treated with cisplatin, and the two main DNA lesions formed, intrastrand adducts and interstrand cross-links, were quantitated before and after repair incubation. This quantitation was done for total genomic lesions and at the level of individual genes. In the overall genome, the initial frequency of both cisplatin lesions assayed was higher in the parental than in the derivative resistant cell lines. Nonetheless, the total genomic repair of each of these lesions was not increased in the resistant cells. These differences in initial lesion frequency between parental and resistant cell lines were not observed at the gene level. Resistant and parental cells had similar initial frequencies of intrastrand adducts and interstrand cross-links in the dihydrofolate reductase (DHFR) gene and in several other genes after cisplatin treatment of the cells. There was no increase in the repair efficiency of intrastrand adducts in the DHFR gene in resistant cell lines compared with the parental partners. However, a marked and consistent repair difference between parental and resistant cells was observed for the gene-specific repair of cisplatin interstrand cross-links. DNA interstrand cross-links were removed from three genes, the DHFR, multidrug resistance (MDR1), and delta-globin genes, much more efficiently in the resistant cell lines than in the parental cell lines. Our findings suggest that acquired cellular resistance to cisplatin may be associated with increased gene-specific DNA repair efficiency of a specific lesion, the interstrand cross-link.
Mol Cell Biol 1992 Sep
PMID:Increased gene-specific repair of cisplatin interstrand cross-links in cisplatin-resistant human ovarian cancer cell lines. 138 Jun 46

An understanding of replication initiation in mammalian cells has been hampered by the lack of mutations and/or inhibitors that arrest cells just prior to entry into the S period. The plant amino acid mimosine has recently been suggested to inhibit cells at a regulatory step in late G1. We have examined the effects of mimosine on cell cycle traverse in the mimosine [corrected]-resistant CHO cell line CHOC 400. When administered to cultures for 14 h after reversal of a G0 block, the drug appears to arrest the population at the G1/S boundary, and upon its removal cells enter the S phase in a synchronous wave. However, when methotrexate is administered to an actively dividing asynchronous culture, cells are arrested not only at the G1/S interface but also in early and middle S phase. Most interestingly, two-dimensional gel analysis of replication intermediates in the initiation locus of the amplified dihydrofolate reductase domain suggests that mimosine may actually inhibit initiation. Thus, this drug represents a new class of inhibitors that may open a window on regulatory events occurring at individual origins of replication.
Mol Cell Biol 1992 Oct
PMID:The plant amino acid mimosine may inhibit initiation at origins of replication in Chinese hamster cells. 140 27

A rapid colorimetric assay for the detection of DNA from Plasmodium falciparum malaria is described, allowing direct sequencing of amplified fragments in the positive samples. The method is based on amplification by the polymerase chain reaction (PCR), with incorporation of biotin and a lac operator sequence in the amplified target DNA. The PCR product was immobilized on streptavidin-coupled magnetic beads, and detected by the specific binding of an Escherichia coli lac repressor beta-galactosidase fusion protein. Positive samples were subsequently treated with alkali to generate single stranded templates, which were used for solid phase genomic sequencing. As targets for amplification and sequencing we selected a region of the gene for the antigen Pf155/RESA and a region of the parasite dihydrofolate reductase gene (PfDHFR/TS). We show here that both of these gene targets can be used for specific detection of P. falciparum in patient blood samples. Genomic sequencing of five patient isolates revealed no variation in the Pf155/RESA gene fragment. In a comparison of this sequence with conserved protein domains, a marked similarity to the src homology region 3 was detected. A point mutation was found in the PfDHFR/TS gene fragment of one of the clinical samples, replacing Ser108 with Asn. This mutation has earlier been described in pyrimethamine and cycloguanile-resistant strains of P. falciparum.
Mol Cell Probes 1992 Jun
PMID:Colorimetric detection of Plasmodium falciparum and direct sequencing of amplified gene fragments using a solid phase method. 140 28


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