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)

This report describes the complete nucleotide sequence of the genome of herpesvirus saimiri, the prototype of gammaherpesvirus subgroup 2 (rhadinoviruses). The unique low-G + C-content DNA region has 112,930 bp with an average base composition of 34.5% G + C and is flanked by about 35 noncoding high-G + C-content DNA repeats of 1,444 bp (70.8% G + C) in tandem orientation. We identified 76 major open reading frames and a set of seven U-RNA genes for a total of 83 potential genes. The genes are closely arranged, with only a few regions of sizable noncoding sequences. For 60 of the predicted proteins, homologous sequences are found in other herpesviruses. Genes conserved between herpesvirus saimiri and Epstein-Barr virus (gammaherpesvirus subgroup 1) show that their genomes are generally collinear, although conserved gene blocks are separated by unique genes that appear to determine the particular phenotype of these viruses. Several deduced protein sequences of herpesvirus saimiri without counterparts in most of the other sequenced herpesviruses exhibited significant homology with cellular proteins of known function. These include thymidylate synthase, dihydrofolate reductase, complement control proteins, the cell surface antigen CD59, cyclins, and G protein-coupled receptors. Searching for functional protein motifs revealed that the virus may encode a cytosine-specific methylase and a tyrosine-specific protein kinase. Several herpesvirus saimiri genes are potential candidates to cooperate with the gene for saimiri transformation-associated protein of subgroup A (STP-A) in T-lymphocyte growth stimulation.
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PMID:Primary structure of the herpesvirus saimiri genome. 132 Dec 87

A study of the repair of DNA damage in the dihydrofolate reductase (dhfr) gene of SV40-transformed human fibroblasts after treatment with 8-methoxypsoralen (8MOP) and UVA is described. 8MOP+UVA-induced cross-links in the dhfr gene were completely repaired by 12 h in one normal and one Fanconi's anaemia (FA) group A cell line. In contrast, approximately 35% of cross-links in an episomally maintained Epstein--Barr virus derived plasmid remained unrepaired even after 48 h. Cross-linkable monoadducts in the dhfr gene were repaired more slowly than cross-links, and there was no detectable repair of cross-linkable monoadducts in the plasmid. Thus the ability of a cell to repair 8MOP+UVA-induced cross-links or cross-linkable monoadducts in an episome does not reflect its capacity to repair such lesions in genomic DNA.
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PMID:Repair of 8-methoxypsoralen + UVA-induced damage in specific sequences in chromosomal and episomal DNA in human cells. 254 11

The enzyme dihydrofolate reductase (DHFR) is found ubiquitously in both prokaryotes and eukaryotes. It is essential for de novo synthesis of purines and of deoxythymidine monophosphate for DNA synthesis. Among viruses, however, only the T-even and T5 bacteriophage have been found to encode their own DHFR. In this study a gene for DHFR was found in a specific subgroup of the gamma or lymphotropic class of herpesviruses. DNA sequences for DHFR were found in herpesvirus saimiri and herpesvirus ateles but not in Epstein-Barr virus, Marek's disease virus, herpes simplex virus, varicella-zoster virus, herpesvirus tamarinus, or human cytomegalovirus. The predicted sequence of herpesvirus saimiri DHFR is 186 amino acids in length, the same length as human, murine, and bovine DHFR. The human and herpesvirus saimiri DHFRs share 83 percent positional identity in amino acid sequence. The herpesvirus saimiri DHFR gene is devoid of intron sequences, suggesting that it was acquired by some process involving reverse transcription. This is to our knowledge the first example of a mammalian virus with a gene for DHFR.
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PMID:A gene for dihydrofolate reductase in a herpesvirus. 283 Jun 73

Werner's syndrome (WS) is a human segmental progerioid disorder with an autosomal recessive pattern of inheritance. Patients with WS exhibit a number of symptoms resembling a premature aging phenotype. We have examined the fine structure of the DNA repair of UV-induced cyclobutane pyrimidine dimers in Epstein-Barr virus (EBV)-transformed WS lymphoblastoid cell lines and in a primary WS fibroblast cell line. The repair was measured at the level of the gene and also in the general genome. Gene-specific and strand-specific DNA repair was measured in the actively transcribed genes dihydrofolate reductase (DHFR), c-myc, and p53, and in the transcriptionally inactive regions, delta globin and the X-linked 754 domain. Both gene-specific repair and strand-specific repair were deficient in the transformed WS lymphoblastoid cell lines compared to normal controls. In normal cells, repair in the transcribed strand was 25 (4 h), 43 (8 h), and 72% (24 h); in the WS cells on average, repair in the transcribed strand was 18 (4 h), 27 (8 h), and 44% (24 h). However, in the primary WS fibroblast cell line, we found a pattern of preferential gene repair which was similar to that in normal human cells. In contrast to cells from patients with the gene-specific repair deficient disease Cockayne's syndrome, which show greatly delayed RNA synthesis recovery after UV irradiation, the WS cells had normal recovery of RNA synthesis. The DNA repair results differ for the different cell types, and our findings thus do not establish a general DNA repair phenotype for WS cells. The fibroblasts had proficient repair, but in the WS lymphoblasts we find a deficiency in DNA repair which could contribute to the reported hypermutability in these cells. The lymphoblasts are, however, transformed cells, and it raises the concern that biological findings in transformed cells may not reflect the situation in primary cells.
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PMID:DNA repair fine structure in Werner's syndrome cell lines. 861 4

The c-erbB-2 receptor tyrosine kinase is often overexpressed in human tumors, but the functional implications of this phenotype remain unclear. We previously used phosphorylation-specific antibodies to define major differences in c-erbB-2 tyrosine kinase activity between overexpressing human tumor cell lines (Epstein, R. J., Druker, B. J., Roberts, T. M., and Stiles, C. D. (1992) Proc. Natl. Acad. Sci. U. S. A. 89, 10435-10439). Here we extend this approach to define the relationship between c-erbB-2 tyrosine phosphorylation and protein kinase C (PKC)-dependent transmodulation. Phosphorylation-specific antibodies to the juxtamembrane PKC site Thr686 recognize tyrosine-dephosphorylated wild-type c-erbB-2 following G8/DHFR 3T3 cell treatment with PKC agonists. B104-1-1 cells transformed by activated c-erbB-2 express a subset of tyrosine-phosphorylated receptors that are homologously phosphorylated on Thr686, indicating that Thr686 phosphorylation alone is insufficient to abrogate receptor tyrosine phosphorylation. Similarly, the c-erbB-2-overexpressing human cancer cell lines SK-Ov-3 and BT-474 express constitutively Thr686-phosphorylated receptors. SK-Ov-3 cells express predominantly kinase-inactive c-erbB-2 that is heavily Thr686-phosphorylated, indicating that Thr686 phosphorylation in this line is heterologous in origin. In contrast, BT-474 cells express constitutively autophosphorylated c-erbB-2 despite Thr686 phosphorylation. These results indicate that Thr686 phosphorylation does not directly abolish c-erbB-2 activity and suggest that such phosphorylation reflects constitutive PKC activity induced by either receptor-activating mutations or heterologous growth factors. The latter possibility suggests in turn that c-erbB-2 interacts in an as yet undefined way with heterologous growth factor receptors in human tumor cells.
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PMID:Human cancer cells exhibit protein kinase C-dependent c-erbB-2 transmodulation that correlates with phosphatase sensitivity and kinase activity. 870 75

Serodiagnosis of Epstein-Barr virus (EBV) infection is currently based on the detection of antibodies to distinct EBV antigens by immunofluorescence and enzyme-linked immunosorbent assay-based tests, or in part on the detection of heterophile antibodies by the Paul-Bunnell-Davidson heterophile assay. In the past few years, the specificity and the sensitivity of serodiagnostic assay systems has been improved considerably by the use of purified recombinant EBV antigens. Screening of EBV-positive sera for antigenic reactivities by immunoprecipitation with extracts of EBV-positive cells revealed a 23-kDa protein (p23) that was recognized by antibodies from all EBV carriers tested. Open reading frame BLRF2 was identified as the coding region for this protein. After cloning and high-level expression of the BLRF2 open reading frame as DHFR fusion protein in Escherichia coli, the recombinant protein was purified to near homogeneity with the help of continuous elution electrophoresis. Sera from both EBV-positive and -negative donors were screened by immunoblot analysis and enzyme-linked immunosorbent assay for IgM and IgG antibodies against the EBV-encoded protein p23. Since anti-p23 antibodies were not detectable in 30 of 30 EBV-negative sera, and 294 of 302 EBV-positive sera had either IgM and/or IgG antibody responses to this protein, recombinant p23 seems to be a useful diagnostic marker for EBV-infection.
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PMID:Expression and purification of an Epstein-Barr virus encoded 23-kDa protein and characterization of its immunological properties. 891 25

Two-dimensional heteronuclear (1H-15N) nuclear magnetic relaxation studies of dihydrofolate reductase (DHFR) from Escherichia coli have demonstrated that glycine-121 which is 19 A from the catalytic center of the enzyme has large-amplitude backbone motions on the nanosecond time scale [Epstein, D. M., Benkovic, S. J., and Wright, P. E. (1995) Biochemistry 34, 11037-11048]. In order to probe the dynamic-function relationships of this residue, we constructed a mutant enzyme in which this glycine was changed to valine. Equilibrium binding studies indicated that the Val-121 mutant retained wild-type binding properties with respect to dihydrofolate and tetrahydrofolate; however, binding to NADPH and NADP+ was decreased by 40-fold and 2-fold, respectively, relative to wild-type DHFR. Single-turnover experiments indicated that hydride transfer was reduced by 200-fold to a rate of 1.3 s-1 and was the rate-limiting step in the steady state. Interestingly, pre-steady-state kinetic analysis of the Val-121 mutant revealed a conformational change which preceded chemistry that occurred at a rate of 3.5 s-1. If this step exists in the kinetic mechanism of the wild-type enzyme, then it would be predicted to occur at a rate of approximately 2000 s-1. Glycine-121 was also changed to alanine, serine, leucine, and proline. While the Ala-121 and Ser-121 mutants behaved similar to wild-type DHFR, the Leu-121 and Pro-121 mutants behaved like Val-121 DHFR in that hydride transfer was the rate-limiting step in the steady state and a conformational change preceding chemistry was observed. Finally, insertion of a glycine or valine between amino acids 121 and 122 produced mutant enzymes with properties similar to wild-type or Val-121 DHFRs, respectively. Taken together, these results provide compelling evidence for dynamic coupling of a remote residue to kinetic events at the active site of DHFR.
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PMID:Evidence for a functional role of the dynamics of glycine-121 of Escherichia coli dihydrofolate reductase obtained from kinetic analysis of a site-directed mutant. 939 9

Analysis of the dihydrofolate reductase (DHFR) complex with folate by two-dimensional heteronuclear (1H-15N) nuclear magnetic relaxation revealed that isolated residues exhibit diverse backbone fluctuations on the nanosecond to picosecond time scale [Epstein, D. M., Benkovic, S. J., and Wright, P. E. (1995) Biochemistry 34, 11037-11048]. These dynamical features may be significant in forming the Michaelis complex. Of these residues, glycine 121 displays large-amplitude backbone motions on the nanosecond time scale. This amino acid, strictly conserved for prokaryotic DHFRs, is located at the center of the betaF-betaG loop. To investigate the catalytic importance of this residue, we report the effects of Gly121 deletion and glycine insertion into the modified betaF-betaG loop. Relative to wild type, deletion of Gly121 dramatically decreases the rate of hydride transfer 550-fold and the strength of cofactor binding 20-fold for NADPH and 7-fold for NADP+. Furthermore, DeltaG121 DHFR requires conformational changes dependent on the initial binary complex to attain the Michaelis complex poised for hydride transfer. Surprisingly, the insertion mutants displayed a significant decrease in both substrate and cofactor binding. The introduction of glycine into the modified betaF-betaG loop, however, generally eliminated conformational changes required by DeltaG121 DHFR to attain the Michaelis complex. Taken together, these results suggest that the catalytic role for the betaF-betaG loop includes formation of liganded complexes and proper orientation of substrate and cofactor. Through a transient interaction with the Met20 loop, alterations of the betaF-betaG loop can orchestrate proximal and distal effects on binding and catalysis that implicate a variety of enzyme conformations participating in the catalytic cycle.
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PMID:Deletion of a highly motional residue affects formation of the Michaelis complex for Escherichia coli dihydrofolate reductase. 957 47

Herpesvirus gene expression can be classified into four distinct kinetic stages: latent, immediate early, early, and late. Here we characterize the kinetic class of a group of 16 Kaposi's sarcoma-associated herpesvirus (KSHV)/human herpesvirus 8 genes in a cultured primary effusion cell line and examine the expression of a subset of these genes in KS biopsies. Expression of two latent genes, LANA and vFLIP, was constitutive and was not induced by chemicals that induce the lytic cycle in primary effusion lymphoma (PEL) cell lines. An immediate-early gene, Rta (open reading frame 50 [ORF50]), was induced within 4 h of the addition of n-butyrate, and its 3.6-kb mRNA was resistant to inhibition by cycloheximide. Early genes, including K3 and K5 that are homologues of the "immediate-early" gene of bovine herpesvirus 4, K8 that is a positional homologue of Epstein-Barr virus BZLF1, vMIP II, vIL-6, and polyadenylated nuclear (PAN) RNA, appeared 8 to 13 h after chemical induction. A second group of early genes that were slightly delayed in their appearance included viral DHFR, thymidylate synthase, vMIP I, G protein-coupled receptor, K12, vBcl2, and a lytic transcript that overlapped LANA. The transcript of sVCA (ORF65), a late gene whose expression was abolished by Phosphonoacetic acid, an inhibitor of KSHV DNA replication, did not appear until 30 h after induction. Single-cell assays indicated that the induction of lytic cycle transcripts resulted from the recruitment of additional cells into the lytic cycle. In situ hybridization of KS biopsies showed that about 3% of spindle-shaped tumor cells expressed Rta, ORF K8, vIL-6, vMIP I, vBcl-2, PAN RNA, and sVCA. Our study shows that several KSHV-encoded homologues of cellular cytokines, chemokines, and antiapoptotic factors are expressed during the viral lytic cycle in PEL cell lines and in KS biopsies. The lytic cycle of KSHV, probably under the initial control of the KSHV/Rta gene, may directly contribute to tumor pathogenesis.
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PMID:Kinetics of Kaposi's sarcoma-associated herpesvirus gene expression. 997 6

Kaposi's sarcoma-associated herpesvirus (KSHV), the most recently discovered human tumour virus, is the causative agent of Kaposi's sarcoma, primary effusion lymphoma and some forms of Castleman's disease. KSHV is a rhadinovirus, and like other rhadinoviruses, it has an extensive array of regulatory genes obtained from the host cell genome. These pirated KSHV proteins include homologues to cellular CD21, three different beta-chemokines, IL-6, BCL-2, several different interferon regulatory factor homologues, Fas-ligand ICE inhibitory protein (FLIP), cyclin D and a G-protein-coupled receptor, as well as DNA synthetic enzymes including thymidylate synthase, dihydrofolate reductase, DNA polymerase, thymidine kinase and ribonucleotide reductases. Despite marked differences between KSHV and Epstein-Barr virus, both viruses target many of the same cellular pathways, but use different strategies to achieve the same effects. KSHV proteins have been identified which inhibit cell-cycle regulation checkpoints, apoptosis control mechanisms and the immune response regulatory machinery. Inhibition of these cellular regulatory networks app ears to be a defensive means of allowing the virus to escape from innate antiviral immune responses. However, due to the overlapping nature of innate immune and tumour-suppressor pathways, inhibition of these regulatory networks can lead to unregulated cell proliferation and may contribute to virus-induced tumorigenesis.
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PMID:Molecular virology of Kaposi's sarcoma-associated herpesvirus. 1131 14


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