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)

The retroviral gene transfer system is a powerful tool for somatic gene therapy. A retroviral stock with a high viral titer and lacking replication-competent virus (RCV) is desirable for this type of gene transfer. To fulfill these requirements, we made a new packaging cell line, designated ampli-GPE. To reduce the homology between proviral DNA in the packaging cell and retroviral vector, the gag-pol and env genes of Moloney murine leukemia virus were separated onto two different plasmids, pGP-KV and pENV-KV, respectively, in which the 5' long terminal repeat and the 3' long terminal repeat had been replaced by the mouse metallothionein I promoter or the human beta-globin gene containing the polyadenylation site as control units for the gag-pol and env genes. In addition, these plasmids contained 69% of the bovine papillomavirus gene for gene amplification to obtain production of virus at a high titer. NIH 3T3 clones containing approximately 20 to 50 copies of the gag-pol and env genes were selected and designated ampli-GPE. When ampli-GPE was transfected with the N2 vector or pZipNeoSV(DHFR) derived from pZipNeoSV(X)1, we established clones producing titers of 5 x 10(6) and 1 x 10(6) CFU/ml, respectively. There was no sign of RCV generation in any virus-producing cells from ampli-GPE. However, virus-producing cells derived from psi 2 cells transfected with N2 did generate RCV. Thus, we showed that ampli-GPE, possessing the minimum complement of proviral genes, has potential for the development of a gene transfer system.
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PMID:A new retrovirus packaging cell for gene transfer constructed from amplified long terminal repeat-free chimeric proviral genes. 131 79

DNA polymerase beta (beta-pol) and its mRNA are maintained at constitutive levels during the cell cycle and during stages of cell growth in culture. To study biological consequences of variations in the level of this DNA repair enzyme and/or its mRNA, we prepared expression vectors in which cDNA for human beta-pol is inserted under the control of a metallothionein promoter (pMT) in the sense and antisense orientation, respectively, and these vectors then were used for stable transformation of mouse 3T3 cells. Vectors also contained the mouse DHFR gene, such that culture of transformants in medium with increasing concentrations of methotrexate resulted in amplification of inserted DNA. The levels of sense and antisense transcripts are strongly increased by culture of transformants in medium with 65 microM Zn2+, although some expression is detected even without Zn2+ induction. After five days of induction, the beta-pol level was about threefold higher in sense cells and about 10-fold lower in antisense cells than in parallel cultures without induction. The antisense line has a threefold increased cell doubling time in the presence of 65 microM Zn2+ compared with the absence of Zn2+. Zn2+ (65 microM) induction for the sense line results in normal growth for the first three days and, thereafter, a complete cessation of growth. Yet, these blocked cells remain fully viable. The results indicate that sudden deregulation of beta-pol expression alters cell growth in mouse 3T3 cells.
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PMID:Deregulation of DNA polymerase beta by sense and antisense RNA expression in mouse 3T3 cells alters cell growth. 169 88

The bifunctional thymidylate synthase-dihydrofolate reductase (TS-DHFR) of Leishmania major has been cloned and expressed in Escherichia coli and Saccharomyces cerevisiae. The strategy involved placing the entire 1560-bp coding sequence into a parent cloning plasmid that was designed to permit introduction of unique restriction sites at the 5'- and 3'-ends. In this manner, the entire coding sequence could be easily subcloned into a variety of expression vectors. High levels of TS-DHFR gene expression were driven by tac, pL and T7 RNA pol promoters in E. coli, and the GAPDH-ADH-2 promoter in S. cerevisiae. L. major TS-DHFR also complemented TS deficiency in E. coli. In E. coli, the protein accumulated to very high levels, but most was present as inactive inclusion bodies. Nevertheless, substantial amounts were soluble; up to 2% of the soluble protein was catalytically active TS-DHFR. In the yeast systems, essentially all of the bifunctional protein was soluble and catalytically active, and crude extracts contained about 100-fold more enzyme than do extracts from wild-type L. major. The expressed TS-DHFR from yeast and E. coli was purified to homogeneity by methotrexate-Sepharose affinity chromatography. About 8.5 mg of homogeneous, catalytically active protein is obtained from a 1-L culture of yeast, and 1.5 mg was obtained from 1 L of E. coli culture. A 200-L fermentation of the yeast expression system yielded a crude extract containing over 4 g of TS-DHFR.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Heterologous expression of the bifunctional thymidylate synthase-dihydrofolate reductase from Leishmania major. 284 73

DNA fragments generated by Bam HI restriction endonuclease digestion of the provirus of bovine leukosis virus (BLV) was recloned in several plasmids. Recombinant plasmids containing X-region, env gene and a part of pol gene were prepared in pBR322, and in a plasmid containing promotor PR. Fragments env gene and a part of pol gene inserted were also into the pSV2-dhfr plasmid which has the both bacterial and eukaryotic promotors together with the gene for folic acid reductase. The expression possibility of these inserted BLV sequences either in mammalian cells after transfection or in bacteria is now tested.
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PMID:Bovine leukosis virus: recloning of specific DNA fragments. 302 38

The nucleolus is a unique structural component of interphase nuclei where the ribosomal genes, trans-cribed by RNA polymerase I (RNA pol I), are organized. In the present study, the repair of UV-induced photolesions was investigated in the ribosomal DNA (rDNA) in relation to RNA pol I transcription. We used hamster cells because their repair phenotype permits the separate analysis of the major photo-products induced by UV light. Immunofluorescent labeling of UV-induced DNA repair and transcription sites showed that the nucleolar regions were defic-ient in DNA repair despite the presence of abundant RNA pol I transcription foci. Immunological staining indicated that various NER proteins, including TFIIH (subunits p62 and p89), p53, Gadd 45 and prolifer-ating cell nuclear antigen are all enriched in the nuclei but distinctly absent in nucleoli. This lack of enrichment of NER factors in the nucleolus may be responsible for the inefficient repair of photo-products in the rDNA. UV irradiation generates two major photoproducts, the cyclobutane pyrimidine dimers (CPDs) and the 6-4 photoproducts (6-4 PPs). The repair kinetics of these two lesions were assessed simultaneously by the immunological isolation of bromodeoxyuridine (BudR) containing excision repair patches using an antibody to BudR. We found that the repair of the photolesions was less efficient in the rDNA compared to that of the endo-genous housekeeping gene, dihydrofolate reductase (DHFR). Gene specific repair of each of these two photoproducts was then measured separately in the rDNA and in the DHFR gene, which is transcribed by RNA pol II. The removal of CPDs was deficient in the rDNA as compared to the DHFR gene. On the contrary, 6-4 PPs were removed efficiently from the rDNA although somewhat slower than from the DHFR gene. The relatively efficient repair of 6-4 PPs in the rDNA is consistent with the notion that the 6-4 PPs are repaired efficiently in different genomic regions by the global genome repair pathway.
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PMID:DNA repair of pyrimidine dimers and 6-4 photoproducts in the ribosomal DNA. 1035 80

The 180-amino acid core of the TATA-binding protein (TBPcore) is conserved from Archae bacteria to man. Vertebrate TBPs contain, in addition, a large and highly conserved N-terminal region that is not found in other phyla. We have generated a line of mice in which the tbp allele is replaced with a version, tbp(Delta N), which lacks 111 of 135 N-terminal amino acid residues. Most tbp(Delta N/Delta N) fetuses die in midgestation. To test whether a disruption of general cellular processes contributed to this fetal loss, primary fibroblast cultures were established from +/+, Delta N/+, and Delta N/Delta N fetuses. The cultures exhibited no genotype-dependent differences in proliferation or in expression of the proliferative markers dihydrofolate reductase (DHFR) mRNA (S phase-specific) and cdc25B mRNA (G(2)-specific). The mutation had no effect on transcription initiation site fidelity by either RNA polymerase II (pol II) or pol III. Moreover, the mutation did not cause differences in levels of U6 RNA, a pol III-dependent component of the splicing machinery, in mRNA splicing efficiency, in expression of housekeeping genes from either TATA-containing or TATA-less promoters, or in global gene expression. Our results indicated that general eukaryotic cell functions are unaffected by deletion of these vertebrate-specific sequences from TBP. Thus, all activities of this polypeptide domain must either be compensated for by redundant activities or be restricted to situations that are not represented by primary fibroblasts.
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PMID:Fundamental cellular processes do not require vertebrate-specific sequences within the TATA-binding protein. 1247 Oct 23

Chemical shift data from the BiomagResDataBank and conformational data derived from the protein data bank have been correlated in order to explore the conformational dependence of side chain (13)C resonance shifts. Consistent with predictions based on steric compression, upfield shifts for Cgamma resonances of Thr, Val, Ile, Leu, Met, Arg, Lys, Glu, and Gln residues correlate with both the number of heavy atom (nonproton) gamma-substituents and with gauche conformational orientations of gamma-substituents. The (13)C shift/conformation correlations are most apparent for Cgamma carbons but also can be observed at positions further from the backbone. Intraresidue steric conflict leads to a correlation between upfield-shifted side chain (13)C resonances and statistically lower probabilities in surveys of protein side chain conformation. Illustrative applications to the DNA pol lambda lyase domain and to dihydrofolate reductase are discussed. In the latter case, (13)C shift analysis indicates that the conformation of the remote residue V119 on the betaF-betaG loop is correlated with the redox state of the bound pyridine nucleotide cofactor, providing one basis for discrimination between substrate and product. It is anticipated that (13)C shift data for protein sidechains can provide a useful basis for the analysis of conformational changes even in large, deuterated proteins. Additionally, the large dependence of the leucine methyl shift difference, deltaCdelta1-deltaCdelta2, on both chi1 and chi2 is sufficient to allow this parameter to be used as a restraint in structure calculations if stereospecific assignment data are available.
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PMID:Dependence of amino acid side chain 13C shifts on dihedral angle: application to conformational analysis. 1865 54