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)

Is the enzymatic machinery for DNA precursor biosynthesis linked to the DNA replication apparatus? To identify intermolecular associations among deoxyribonucleotide biosynthetic enzymes and to ask whether these enzymes are linked to replication proteins, we analyzed radiolabeled T4 bacteriophage proteins that bind specifically to a column of immobilized T4 deoxycytidylate hydroxymethylase. More than a dozen T4 proteins and a few Escherichia coli proteins are adsorbed specifically by this column. Several of the T4 proteins were identified by two-dimensional gel electrophoresis and radioautography. These include five enzymes involved in DNA precursor biosynthesis, dCMP hydroxymethylase, thymidylate synthase, dihydrofolate reductase, dCTPase-dUTPase, and ribonucleotide reductase large and small subunits, plus several proteins of DNA metabolism and replication. Analysis of extracts of cells infected with phage amber mutants defective in specific proteins suggested a specific association involving thymidylate synthase and the gene 32 single-strand DNA-binding protein.
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PMID:Specific associations of T4 bacteriophage proteins with immobilized deoxycytidylate hydroxymethylase. 156 1

The type IV plasmid-mediated dihydrofolate reductase (DHFR), from a clinical strain of Escherichia coli isolated in South India, was prepared from a transconjugant containing the original clinical plasmid, E. coli J62-2 (pUK1123), and from E. coli C600 (pUK1150) containing a 2.6-kb HindIII fragment of pUK1123 cloned into plasmid pBR322. Both preparations were purified by methotrexate affinity chromatography. Automatic amino-acid sequencing of the N-terminal of the purified type IV enzyme from both sources gave an identical sequence which was clearly distinct from other plasmid-mediated trimethoprim-resistant DHFRs. The type IV DHFR showed most homology with the endogenous, chromosomally-encoded E. coli enzyme. Amino-acid sequence analysis also showed that the type IV enzyme preparation from E. coli J62-2 harbouring the original clinical plasmid, pUK1123, also contained the E. coli DNA-binding protein NS1. Analysis by polyacrylamide gel electrophoresis suggested that the type IV enzyme, in its native form, consists of a DHFR of Mr 33,000 coupled to a DNA-binding protein.
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PMID:N-terminal amino-acid sequence and subunit structure of the type IV trimethoprim-resistant plasmid-encoded dihydrofolate reductase. 219 14

Ltk- cells were transfected with a plasmid containing the entire domain of glycoprotein C (gC), a true gamma or gamma 2 gene of herpes simplex virus 1 (HSV-1) and the methotrexate-resistant mouse dihydrofolate reductase mutant gene. The resulting methotrexate-resistant cell line was cloned; of the 39 clonal lines tested only 1, L3153(28), expressed gC after infection with HSV-1(MP), a gC- mutant, and none expressed gC constitutively. The induction of gC was optimal at multiplicities ranging between 0.5 and 2 PFU per cell, and the quantities produced were equivalent to or higher than those made by methotrexate-resistant gC- L cells infected with wild-type (gC+) virus. The gC gene resident in the L3153(28) cells was regulated as a beta gene inasmuch as the amounts of gC made in infected L3153(28) cells exposed to concentrations of phosphonoacetate that inhibited viral DNA synthesis were higher than those made in the absence of the drug, gC was induced at both permissive and nonpermissive temperatures by the DNA- mutant tsHA1 carrying a lesion in the gene specifying the major DNA-binding protein and which does not express gamma 2 genes at the nonpermissive temperature, and gC was induced only at the permissive temperature in cells infected with ts502 containing a mutation in the alpha 4 gene. The gC induced in L3153(28) cells was made earlier and processed faster to the mature form than that induced in a gC- clone of methotrexate-resistant cells infected with wild-type virus. Unlike virus stocks made in gC- cells, HSV-1(MP) made in L3153(28) cells was susceptible to neutralization by anti-gC monoclonal antibody.
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PMID:Expression and regulation of glycoprotein C gene of herpes simplex virus 1 resident in a clonal L-cell line. 300 54

The transcription factor E2F activates the expression of multiple genes involved in cell proliferation, such as c-myc and the dihydrofolate reductase gene. Regulation of E2F involves its interactions with other cellular proteins, including the retinoblastoma protein (Rb), the Rb-related protein p107, cyclin A, and cdk2. We undertook a detailed analysis of E2F DNA-binding activities and their cell cycle behavior in primary human T cells. Three E2F DNA-binding activities were identified in resting (G0) T cells with mobilities in gel shift assays distinct from those of previously defined E2F complexes. One of these activities was found to be a novel, less abundant, Rb-E2F complex. The most prominent E2F activity in resting T cells (termed complex X) was abundant in both G0 and G1 but disappeared as cells entered S phase, suggesting a possible role in negatively regulating E2F function. Complex X could be dissociated by adenovirus E1A with a requirement for an intact E1A conserved region 2. However, X failed to react with a variety of antibodies against Rb or p107, implicating the involvement of an E1A-binding protein other than Rb or p107. In addition to these novel E2F complexes, three distinct forms of unbound (free) E2F were resolved in gel shift experiments. These species showed different cell cycle kinetics. UV cross-linking experiments suggested that a distinct E2F DNA-binding protein is uniquely associated with the S-phase p107 complex and is not associated with Rb. Together, these results suggest that E2F consists of multiple, biochemically distinct DNA-binding proteins which function at different points in the cell cycle.
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PMID:Cell cycle analysis of E2F in primary human T cells reveals novel E2F complexes and biochemically distinct forms of free E2F. 832 Dec 4

After T4 bacteriophage infection of Escherichia coli, the enzymes of deoxyribonucleoside triphosphate biosynthesis form a multienzyme complex that we call T4 deoxyribonucleoside triphosphate (dNTP) synthetase. At least eight phage-coded enzymes and two enzymes of host origin are found in this 1.5-mDa complex. The complex may shuttle dNTPs to DNA replication sites, because replication draws from small pools, which are probably highly localized. Several specific protein-protein contacts within the complex are described in this paper. We have studied protein-protein interactions in the complex by immobilizing individual enzymes and identifying radiolabeled T4 proteins that are retained by columns of these respective affinity ligands. Elsewhere we have described interactions involving three T4 enzymes found in the complex. In this paper we describe similar analysis of five more proteins: dihydrofolate reductase, dCTPase-dUTPase, deoxyribonucleoside monophosphokinase, ribonucleotide reductase, and E. coli nucleoside diphosphokinase,. All eight proteins analyzed to date retain single-strand DNA-binding protein (gp32), the product of T4 gene 32. At least one T4 protein, thymidylate synthase, binds directly to gp32, as shown by affinity chromatographic analysis of the two purified proteins. Among its several roles, gp32 stabilizes single-strand template DNA ahead of a replicating DNA polymerase. Our data suggest a model in which dNTP synthetase complexes, probably more than one per growing DNA chain, are drawn to replication forks via their affinity for gp32 and hence are localized so as to produce dNTPs at their sites of utilization, immediately ahead of growing DNA 3' termini.
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PMID:T4 phage gene 32 protein as a candidate organizing factor for the deoxyribonucleoside triphosphate synthetase complex. 862 61

The current paradigm for elucidating the molecular etiology of cancers relies on the interrogation of small numbers of genes, which limits the scope of investigation. Emerging second-generation massively parallel DNA sequencing technologies have enabled more precise definition of the cancer genome on a global scale. We examined the genome of a human primary malignant pleural mesothelioma (MPM) tumor and matched normal tissue by using a combination of sequencing-by-synthesis and pyrosequencing methodologies to a 9.6X depth of coverage. Read density analysis uncovered significant aneuploidy and numerous rearrangements. Method-dependent informatics rules, which combined the results of different sequencing platforms, were developed to identify and validate candidate mutations of multiple types. Many more tumor-specific rearrangements than point mutations were uncovered at this depth of sequencing, resulting in novel, large-scale, inter- and intra-chromosomal deletions, inversions, and translocations. Nearly all candidate point mutations appeared to be previously unknown SNPs. Thirty tumor-specific fusions/translocations were independently validated with PCR and Sanger sequencing. Of these, 15 represented disrupted gene-encoding regions, including kinases, transcription factors, and growth factors. One large deletion in DPP10 resulted in altered transcription and expression of DPP10 transcripts in a set of 53 additional MPM tumors correlated with survival. Additionally, three point mutations were observed in the coding regions of NKX6-2, a transcription regulator, and NFRKB, a DNA-binding protein involved in modulating NFKB1. Several regions containing genes such as PCBD2 and DHFR, which are involved in growth factor signaling and nucleotide synthesis, respectively, were selectively amplified in the tumor. Second-generation sequencing uncovered all types of mutations in this MPM tumor, with DNA rearrangements representing the dominant type.
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PMID:Second generation sequencing of the mesothelioma tumor genome. 2048 25