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)

Polyclonal and monoclonal antibodies were raised against synthetic peptides (or fusion protein) corresponding to cDNA-deduced amino acid sequences unique to the human and mouse retinoic acid (RA) receptor alpha 1 (hRAR-alpha 1 and mRAR-alpha 1, respectively). Two rabbit polyclonal antibodies directed against either the F region fused to DHFR [RP alpha (F)] or the D2 region [RP alpha (D2)] were selected. Using either immunocytochemistry, Western blotting analysis, or immunoprecipitation, they were found to be specific for human and mouse RAR-alpha 1 proteins produced by COS-1 cells transiently transfected with vectors expressing the RAR-alpha 1 cDNA. Three mouse monoclonal antibodies directed against either the F region [(Ab9 alpha (F) and Ab12 alpha (F)] or the A1 region [Ab10 alpha 1(A1)] recognized transiently expressed human and mouse RAR-alpha 1 proteins, when either immunocytochemistry or immunoprecipitation was used. In addition, Ab9 alpha (F) and Ab12 alpha (F), but not Ab10 alpha 1(A1), revealed the RAR-alpha 1 proteins by Western blotting analysis. Ab9 alpha (F) was also able to "supershift" RAR-alpha 1 protein-RARE oligonucleotide probe complexes in gel retardation assays. All these antibodies recognized also the transiently expressed mRAR-alpha 2 isoform, with the exception of Ab10 alpha 1 (A1), which is specific for the A1 region of RAR-alpha 1. These antibodies have enabled us to detect the presence of mRAR-alpha as multiple species in mouse embryo and adult tissue extracts as well as in embryonal carcinoma (EC) cells. Moreover, we found that one of these species (51 kDa) was phosphorylated in EC cells. This phosphorylation was not affected by RA treatment, but appeared to be dependent on the differentiation state of the EC cells.
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PMID:Immunodetection of multiple species of retinoic acid receptor alpha: evidence for phosphorylation. 132 15

The role of eukaryotic initiation factor 2 (eIF-2) phosphorylation in translational control has been demonstrated in vivo by overexpressing variant forms of eIF-2 alpha that are not phosphorylated. COS-1 cells transiently transfected with expression vectors for human eIF-2 alpha contain 10-20-fold more eIF-2 alpha subunit than the endogenous COS cell eIF-2 trimeric complex. Expression of the variant form of eIF-2 alpha, Ser51Asp, where Asp replaces Ser51, causes inhibition of protein synthesis, whereas the Ser48Asp variant does not. When either Ser48 or Ser51 is replaced by Ala, the variants stimulate dihydrofolate reductase synthesis when the eIF-2 alpha kinase, DAI, is activated. In order to elucidate these mechanisms, we have separated eIF-2 trimeric complexes from free overexpressed eIF-2 alpha subunits by fast protein liquid chromatography Superose chromatography. Pulse-labeled cells transfected with wild-type or variant DNAs produced eIF-2 preparations with greater than 10-fold higher specific radioactivity in the alpha-subunit compared to the gamma-subunit, thus demonstrating that the human eIF-2 alpha produced from the plasmids readily exchanges into COS cell eIF-2 complexes. Both wild-type and Ser48Ala variant forms of the free 2 alpha-subunit, further purified by MonoQ chromatography, are poor substrates for the heme-regulated eIF-2 alpha kinase, HRI, but are good substrates for double-stranded RNA-activated inhibitor in vitro; the Ser51Ala variant subunit is not phosphorylated by either kinase. None of the purified free eIF-2 alpha subunits inhibits phosphorylation of eIF-2 in vitro, even at up to 8-fold molar excess. Examination of the extent of eIF-2 alpha phosphorylation in the COS cell eIF-2 complexes by two-dimensional polyacrylamide gel electrophoresis shows that the stimulation of dihydrofolate reductase synthesis by the Ser51Ala variant is most readily explained by failure of eIF-2 to be phosphorylated. Stimulation by the Ser48Ala variant appears to occur by mitigation of the effect of phosphorylation at Ser51 since the double variant, Ser48Ala-Ser51Asp, inhibits protein synthesis less than the single variant Ser51Asp. The evidence argues strongly against there being a second site of phosphorylation involved in translational repression.
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PMID:Stimulation of protein synthesis in COS cells transfected with variants of the alpha-subunit of initiation factor eIF-2. 134 29

A full-length cDNA clone of 4.3 kb encoding the human ATP-citrate lyase enzyme has been isolated by screening a human cDNA library with the recently isolated rat ATP-citrate lyase cDNA clone [Elshourbagy et al. (1990) J. Biol. Chem. 265, 1430]. Nucleic-acid sequence data indicate that the cDNA contains the complete coding region for the enzyme, which is 1105 amino acids in length with a calculated molecular mass of 121,419 Da. Comparison of the human and rat ATP-citrate lyase cDNA sequences reveals 96.3% amino acid identity throughout the entire sequence. Further sequence analysis identified the His765 catalytic phosphorylation site, the ATP-binding site, as well as the CoA binding site. The human ATP-citrate lyase cDNA clone was subcloned into a mammalian expression vector for expression in African green monkey kidney cells (COS) and Chinese hamster ovary cells (CHO) cells. Transfected COS cells expressed detectable levels of an enzymatically active recombinant ATP-citrate lyase enzyme. Stable, amplified expression of ATP-citrate lyase in CHO cells as achieved by using coamplification with dihydrofolate reductase. Resistant cells expressed high levels of enzymatically active ATP-citrate lyase (3 pg/cell/d). Site-specific mutagenesis of His765----Ala diminishes the catalytic activity of the expressed ATP-citrate lyase protein. Since catalysis of ATP-citrate lyase is postulated to involve the formation of phosphohistidine, these results are consistent with the pattern of earlier observations of the significance of the histidine residue in catalysis of the human ATP-citrate lyase.
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PMID:Cloning and expression of a human ATP-citrate lyase cDNA. 137 49

COS cells transfected with plasmids that activate DAI depend on expression of virus-associated I (VAI) RNA to prevent the inhibitory effects of the alpha subunit of eukaryotic initiation factor 2 (eIF-2 alpha) kinase (DAI) and restore the translation of vector-derived dihydrofolate reductase mRNA. This VAI RNA requirement could be completely replaced by reovirus polypeptide sigma 3, consistent with its double-stranded RNA (dsRNA)-binding activity. S4 gene transfection of 293 cells also partially restored adenovirus protein synthesis after infection with the VAI-negative dl331 mutant. In dl331-infected 293 cells, eIF-2 alpha was present mainly in the acidic, phosphorylated form, and trans complementation with polypeptide sigma 3 or VAI RNA decreased the proportion of eIF-2 alpha (P) from approximately 85 to approximately 30%. Activation of DAI by addition of dsRNA to extracts of S4 DNA-transfected COS cells required 10-fold-higher levels of dsRNA than extracts made from cells that were not producing polypeptide sigma 3. In extracts of reovirus-infected mouse L cells, the concentration of dsRNA needed to activate DAI was dependent on the viral serotype used for the infection. Although the proportion of eIF-2 alpha (P) was greater than that in uninfected cells, most of the factor remained in the unphosphorylated form, even at 16 h after infection, consistent with the partial inhibition of host protein synthesis observed with all three viral serotypes. The results indicate that reovirus polypeptide sigma 3 participates in the regulation of protein synthesis by modulating DAI and eIF-2 alpha phosphorylation.
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PMID:Translational stimulation by reovirus polypeptide sigma 3: substitution for VAI RNA and inhibition of phosphorylation of the alpha subunit of eukaryotic initiation factor 2. 143 98

The high affinity receptor for immunoglobulin E (IgE) is a tetrameric structure (alpha beta gamma 2) consisting of non-covalently associated subunits: one IgE-binding alpha chain, one 4-fold membrane spanning beta chain, and two disulfide-linked gamma chains. Here, we have engineered alpha cDNA constructs (alpha trunc) encoding exclusively the leader peptide and the extracellular domain of the alpha subunit. Transfection of human alpha trunc into COS-7 cells resulted in the secretion of soluble IgE-binding polypeptides. By contrast, the polypeptides generated from rat and mouse alpha trunc transfections were sequestered in the endoplasmic reticulum and degraded even though they appeared to fold properly as judged by their capacity to bind IgE. Stable transfectants with human alpha trunc were obtained from a dihydrofolate reductase-deficient Chinese hamster ovary cell line. Several clones secreted substantial amounts (0.1 microgram/ml/10(6) cells) of IgE-binding polypeptides. The dissociation rate of bound IgE from this soluble truncated alpha (kappa-1 = 4.9 x 10(-6) s-1 at 25 degrees C) was characteristic of receptors on intact cells. After treatment with tunicamycin, the transfectants secreted unglycosylated 18-kDa polypeptides which could also bind IgE. These unglycosylated products had a tendency to form dimers and higher oligomers which were resistant to treatment by sodium dodecyl sulfate and reducing agents. These data demonstrate unequivocally that the extracellular domain of the alpha subunit is sufficient to mediate high affinity binding of IgE. Furthermore, posttranslational addition of carbohydrates is not required for proper folding and function of the receptor binding site. The truncated human alpha should be a suitable reagent for crystallographic analysis and for detailed analysis of the receptor binding sites.
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PMID:Characterization of truncated alpha chain products from human, rat, and mouse high affinity receptor for immunoglobulin E. 182 44

A full-length cDNA for the rat liver enzyme tyrosine aminotransferase has been used to construct mammalian expression vectors by recombinant DNA techniques. These vectors, which have employed either a simian virus 40 or a Rous sarcoma virus promoter, were transfected into a variety of nonhepatic mammalian cell lines in culture. Transient expression of tyrosine aminotransferase was readily observed after transfection into monkey COS cells and mouse L cells. Stable clones that express cloned tyrosine aminotransferase have been isolated from mouse L cells, hamster Wg1a fibroblasts, and Chinese hamster ovary (CHO) cells. A vector capable of expressing both tyrosine aminotransferase and dihydrofolate reductase was stimulated to undergo amplification by treatment with methotrexate in a CHO cell line deficient in the latter enzyme. Levels of tyrosine aminotransferase as much as 50-fold higher than typically seen in glucocorticoid-induced hepatoma cells were achieved in some CHO clones by this technique. The tyrosine aminotransferase produced at these highly amplified levels appeared structurally normal and had no major harmful effects on the cells.
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PMID:Expression and amplification of cloned rat liver tyrosine aminotransferase in nonhepatic cells. 196 11

Protein synthesis initiation factor eIF-4D is a relatively abundant protein in mammalian cells and possesses a unique amino acid residue, hypusine. The role of the hypusine modification in eIF-4D function was addressed by studying the function of eIF-4D variants lacking hypusine. The cloned human cDNA encoding eIF-4D was overexpressed in Escherichia coli and a precursor form lacking hypusine was purified. This protein fails to stimulate methionyl-puromycin synthesis in vitro, nor does it significantly inhibit the action of native eIF-4D. Mammalian expression vectors were constructed with the wild-type cDNA and a mutant form in which the codon for lysine-50 (the residue hypusinated) was altered by site-directed mutagenesis to that for arginine. Transient co-transfection of COS-1 cells with the eIF-4D vector and a vector expressing dihydrofolate reductase led to strong synthesis of both eIF-4D and dihydrofolate reductase. This indicates that normal cellular levels of eIF-4D are saturating in these cells and that excess levels of eIF-4D are not detrimental. Cotransfection with the eIF-4D arginine variant caused no effect on dihydrofolate reductase synthesis, in agreement with the in vitro experiments. The inability of the unhypusinated eIF-4D variants to stimulate methionyl-puromycin synthesis in vitro and to affect protein synthesis in vivo strongly suggests that the hypusine modification is required for eIF-4D activity and for its interaction with the 80 S initiation complex in protein synthesis.
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PMID:Protein synthesis initiation factor eIF-4D. Functional comparison of native and unhypusinated forms of the protein. 250 60

A complete cDNA clone of murine interferon-gamma (MuIFN-gamma) was obtained by recombining two appropriate segments from partial cDNA clones originally identified by colony hybridization with rat IFN-gamma chromosomal gene fragments as probes. An expression vector was constructed in which the cDNA was placed under control of the SV40 early promoter. Transient expression of MuIFN-gamma was obtained by transformation of COS-1 cells. Subsequently, this interferon expression unit was linked to a vector containing a dihydrofolate reductase (DHFR) modular gene and used to transform DHFR(-)-CHO cells. Cell clones were selected that constitutively produce an interferon activity which by several criteria was found to be indistinguishable from natural, splenocyte-derived MuIFN-gamma.
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PMID:Molecular cloning of murine interferon gamma (MuIFN-gamma) cDNA and its expression in heterologous mammalian cells. 299 40

We report here for the first time reconstitution and secretion of functionally active antibody in non-lymphoid cells. Expression vectors for the light and the heavy chain of a monoclonal antibody directed against creatine kinase (EC 2.7.3.2) were introduced into COS and CHO Chinese hamster ovary dhfr- cells. Introduction of the expression vectors separately gave rise to immuno-reactive material in the culture supernatants, but only cotransfection of the expression plasmids resulted in secretion of protein with immuno-reactivity against antibodies directed against mouse heavy and light chains as well as specific antigen-binding affinity, as determined by enzyme-linked immunosorbent assay. Secreted kappa and gamma chains from reconstituted antibody were characterized by immunoadsorption and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. In COS cells, reconstituted antibody was transiently secreted; cotransfection of kappa and gamma chain expression plasmids with a dihydrofolate reductase (DHFR)-expression plasmid into CHO dhfr- cells gave rise to stable transformants secreting functionally active antibody.
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PMID:Reconstitution of functionally active antibody directed against creatine kinase from separately expressed heavy and light chains in non-lymphoid cells. 311 10

A mouse hybridoma cell line was isolated which produces monoclonal antibodies (MAbs) of the IgG2b, kappa subtype directed against the tumour-associated marker human placental alkaline phosphatase (hPLAP). The mRNAs coding for the heavy (H) and light (L) chains were cloned as cDNA copies. These genes were then separately inserted into the eukaryotic expression vector pSV23p, under control of the SV40 early promoter. Both genes were introduced with the DEAE-dextran technique in COSI cells, and 72 hr after transfection, 10 ng/ml functional antibodies could be detected in the supernatant of the cells. Permanent CHO cell lines secreting 100 ng/ml functional antibodies were established upon transfection of CHO (dhfr-) cells with the plasmids containing the H and L cDNAs and the plasmid pAdD26SVp-(A)-3 carrying the mouse dihydrofolate reductase (dhfr) gene. A plasmid construction in which we inserted a stop codon-containing sequence behind the hinge region of the H-chain cDNA sequence yielded immuno-competent F(ab')2 molecules upon transfection of COS or CHO cells. Our results indicate that not only lymphoid cells, but also non-lymphoid cells, are capable of synthesis and assembly of immunoglobulin chains that are immunologically fully competent.
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PMID:Expression of functional mouse antibodies directed against the tumour marker human placental alkaline phosphatase in non-lymphoid cells. 316 43


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