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:2.7.7.49 (reverse transcriptase)
31,746 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The RNA-directed DNA polymerase of the primate type-D retrovirus Mason-Pfizer virus was purified using ion-exchange and affinity chromatography, and molecular sieving. The enzyme was shown to have a molecular weight of approx. 80 000 as determined by sedimentation analysis, molecular sieving and sodium dodecyl sulfate polyacrylamide gel electrophoresis. The purified RNA-directed DNA polymerase retained its ability to use a heteropolymeric RNA as a template. The Mason-Pfizer virus RNA-directed DNA polymerase was also characterized as to its divalent cation preference for several synthetic primertemplates and for heteropolymeric RNA. Mg2+ was preferred as its divalent cation for all primer-templates except oligo(dG).poly(rC)m for which it prefers Mn2+. The Mason-Pfizer virus enzyme was also shown to have a pH optimum of 8-8.5 and a temperature optimum of 37-40 degrees C. The stability of the Mason-Pfizer virus RNA-directed DNA polymerase was shown to differ when measured using different primer-templates.
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PMID:Purification and characterization of the RNA-directed DNA polymerase of a primate type-D retrovirus: Mason-Pfizer virus. 677 21

Synchronized mouse cells (JLS-V9) chronically infected with Rauscher murine leukemia virus were used to study virus production, the synthesis of gag and env precursor proteins, and the expression of env protein on the cell surface during the cell cycle. The amount of virus released into the medium by synchronized cells during a 30-min interval was determined by using the XC plaque assay and by measuring reverse transcriptase activity. The results show that virus production occurs during mitosis. Labeling of the cell surface of synchronized cells with 125I or with fluorescein-conjugated antiserum shows that the amount of gp 70env on the cell surface parallels cellular growth. Therefore, the cell cycle-dependent release of virus is not accompanied by similar variations in the amount of viral envelope protein on the cell surface. Immunoprecipitation of cells labeled with [35S]methionine, followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, was used to measure viral protein synthesis during the cell cycle. The rate of synthesis of gag precursor proteins show three maximums corresponding to the G1, middle S, and late S to G2 phases of the cell cycle. The rate of synthesis of env precursor proteins does not change, suggesting that in these cells the synthesis of these two gene products is controlled separately.
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PMID:Retrovirus gene expression during the cell cycle. I. Virus production, synthesis, and expression of viral proteins in Rauscher murine leukemia virus-infected mouse cells. 728 18

Biosynthesis of the activated sulfate donor, adenosine 3'-phosphate 5'-phosphosulfate, involves the sequential action of two enzyme activities: ATP sulfurylase, which catalyzes the formation of adenosine 5'-phosphosulfate (APS) from ATP and free sulfate, and APS kinase, which subsequently phosphorylates APS to produce adenosine 3'-phosphate 5'-phosphosulfate. Oligonucleotide primers were derived from a human infant brain-expressed sequence tag putatively encoding a portion of APS kinase. Using these primers, reverse transcriptase-polymerase chain reaction was performed on mRNA from neonatal normal mice resulting in amplification of a 127-bp DNA fragment. This fragment was subsequently used to screen a mouse brain lambda gt11 cDNA library, yielding a 2.2-kb clone. Primers were designed from the 5'-end of the 2.2-kb clone, and 5'-rapid amplification of cDNA ends was used to obtain the translation start site. Sequence from the overlapping clones was assembled into a 2475-bp composite sequence, which contains a single open reading frame that translates into a 624-deduced amino acid sequence. Northern blots of total RNA from neonatal mice yielded a single message species at approximately 3.3 kb. Southern blot of genomic DNA digested with several restriction enzymes suggested the gene is present as a single copy. Comparison against sequence data bases suggested the composite sequence was a fused sulfurylase-kinase product, since the deduced amino acid sequence showed extensive homology to known separate sequences of both ATP sulfurylase and APS kinase from several sources. The first 199 amino acids corresponded to APS kinase sequence, followed by 37 distinct amino acids, which did not match any known sequence, followed by 388 amino acids that are highly homologous to known ATP sulfurylase sequences. Finally, recombinant enzyme expressed in COS-1 cells exhibited both ATP sulfurylase and APS kinase activity.
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PMID:The isolation and characterization of cDNA encoding the mouse bifunctional ATP sulfurylase-adenosine 5'-phosphosulfate kinase. 749 84

Recently, we purified rat thrombopoietin (TPO) from plasma of irradiated rats (XRP) by measuring its activity that stimulated the production of megakaryocytes from megakaryocyte progenitor cells (CFU-MK) in vitro. We then cloned the cDNAs for rat and human TPO. In this study, we found the production of TPO by hepatocytes isolated with the collagenase perfusion method from both normal and thrombocytopenic rats, by a two-step fractionation of hepatocyte culture medium (CM). Subsequently, CM of rat hepatoma cell lines was screened for the presence of TPO; three cell lines, H4-II-E, McA-RH8994, and HTC, were found to produce TPO. According to the purification procedure for TPO from XRP, TPO was partially purified from 2 L CM of each of three cell lines with a six-step procedure. In the final reverse-phase column, TPO from each cell line was eluted with the same retention time as that from XRP, and the TPO fraction exhibited megakaryocyte colony-stimulating activity (Meg-CSA). TPO-active fraction eluted from the final reverse-phase column was separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), extracted from the gel, and assayed. TPO activity from each cell line was found in the respective molecular weight region, indicating the heterogeneity of the TPO molecule. Using reverse transcriptase-polymerase chain reaction (RT-PCR), we detected the expression of TPO mRNA in hepatocytes, three hepatoma cell lines, normal rat liver, and X-irradiated rat liver. Northern blot analysis showed that TPO mRNA was expressed mainly in liver among the various organs tested. These data demonstrate that TPO is produced by rat hepatocytes and hepatoma cell lines and suggest that liver may be the primary organ that produces TPO.
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PMID:Production of thrombopoietin (TPO) by rat hepatocytes and hepatoma cell lines. 749 68

R82913 and R86183, two derivatives of tetrahydroimidazo[4,5,1-jk][1,4]-benzodiazepin-2(1H)-thione (TIBO), were found to potently and selectively inhibit the replication and cell killing effects of a panel of biologically diverse laboratory and clinical strains of HIV-1. The two compounds exhibited significant activity in all human cell lines tested, as well as in fresh human peripheral blood lymphocytes and macrophages. One of these two compounds (R82913) was found to significantly inhibit the replication of a murine retrovirus (Rauscher murine leukemia virus) in both UV-XC plaque formation and virus yield reduction assays. R86183, despite differing from R82913 only in the positioning of a single chlorine molecule, was not active against the murine retrovirus but was 10-fold more potent in inhibiting HIV-1 replication. Combination antiviral assays with other reverse transcriptase inhibitors, including AZT, ddC, and carbovir, yielded synergistic anti-HIV activity with both TIBO derivatives. Additive to slightly synergistic results were obtained in combinations with ddI and phosphonoformic acid whereas additive to antagonistic activity was detected in combination with dextran sulfate.
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PMID:Differential antiviral activity of two TIBO derivatives against the human immunodeficiency and murine leukemia viruses alone and in combination with other anti-HIV agents. 750 23

Wild-type and several mutant forms of recombinant human immunodeficiency virus type-1 reverse transcriptase were overexpressed as either the p66 or the p51 subunit in a protease-deficient strain of Escherichia coli. Immediately prior to cell lysis, p51 cell paste was mixed with cell paste containing the corresponding overexpressed p66 subunit in a ratio resulting in an excess of the smaller subunit with respect to the larger. During the subsequent chromatography steps stable heterodimer p66/p51 was purified to homogeneity. This protein was characterized by amino acid analysis, denaturing sodium dodecyl sulfate-polyacrylamide gel electrophoresis, analytical gel filtration HPLC, laser desorption mass spectroscopy, and isoelectric focusing. In addition, we were able to obtain crystals of the purified enzyme complexed with a quinazolinone class nonnucleoside inhibitor that diffracted to 3.2 A resolution. A potential application of this expression/purification methodology is the ability to alter specific amino acids residues, by site-directed-mutagenesis, of only one subunit of the RT-dimer.
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PMID:Purification and characterization of HIV-1 reverse transcriptase having a 1:1 ratio of p66 and p51 subunits. 753 52

Mounting experimental evidence suggests that the TAT protein, released from human immunodeficiency virus-1 (HIV-1)-infected inflammatory cells, may genetically reprogram targeted cells within a localized environment to develop highly vascularized tumors of mesenchymal origin. The fibroblast growth factor (FGF) family of polypeptides has gained general acceptance as initiators of angiogenesis and functions as potent mitogens for mesoderm-derived cells. To evaluate a potential biological relationship between TAT and acidic FGF (FGF-1), primary murine embryonic fibroblasts either were transfected with the viral transactivator or were transduced (retrovirally mediated) with a secreted, chimeric form of the human polypeptide growth factor, human stomach tumor/Kaposi's sarcoma (hst/KS)FGF-1. Reverse transcriptase-polymerase chain reaction, Western blotting, in situ immunohistochemical, heparin affinity, DNA synthesis, and transient transfection techniques were used to confirm expression, localization, and functionality of the transgenes. Both transfected and transduced cells constitutively expressing either TAT or (hst/KS)FGF-1 adopted a transformed phenotype, maintained aggressive growth behavior, and demonstrated both induction of FGF-specific phosphotyrosyl proteins and nuclear association of FGF-1 and FGF-1 receptor. Increased levels of endogenous, murine FGF-1 mRNA (reverse transcriptase-polymerase chain reaction) and protein (immunoblot analysis) were apparent in both (hst/KS)FGF-1- and TAT-transformed cells. Medium conditioned by (hst/KS)FGF-1-transduced cells contained steady-state levels of biologically active FGF-1 which exhibited a representative molecular weight. Limited sodium dodecyl sulfate-polyacrylamide gel electrophoretic analysis of the conditioned medium from TAT-transformed cells demonstrated the appearance of FGF-1 as latent, high molecular weight complexes requiring reducing agents to activate full biological activity. Collectively, these results suggest that TAT induces the expression and secretion of FGF-1, which may be potentially relevant to the pathophysiological development of AIDS-Kaposi's sarcoma.
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PMID:The HIV-1 TAT protein induces the expression and extracellular appearance of acidic fibroblast growth factor. 754 39

V511 and V513 are Chinese hamster cell lines with acquired resistance to topoisomerase II (topo II) directed agents. These cell lines were obtained by mutagenizing Chinese hamster V79 cells with N-methyl-N'-nitro-N-nitrosoguanidine and subsequently selecting in etoposide (VP-16). We have previously shown that this resistance is not associated with alterations in drug uptake. To elucidate whether any alterations in the functionally important domains of topo II alpha were associated with VP-16 resistance, we used reverse transcriptase-polymerase chain reaction, single-strand conformational polymorphism analysis, and subsequent sequencing of topo II alpha from V79, V511, and V513 to search for mutations in five major functional domains including the regions of the consensus ATP binding sequences (Motif A and Motif B/dinucleotide binding site), the DNA binding domain, and the 5' and 3' flanking regions of the DNA binding position. The V511 cells showed no mutational changes in these regions. However, the topo II alpha gene from V513 showed a point mutation at nucleotide 2552 that resulted in a glycine-to-aspartate mutation at amino acid position 851 in the 3' flanking region of the DNA binding site. This mutation at amino acid position 851 in V513 cells is associated with reduced VP-16-induced cleavable complex formation demonstrated by potassium-sodium dodecyl sulfate assay and band-depletion analysis. Our results suggest that the mutation at amino acid position 851 may play a role in drug resistance, presumably by interfering with enzyme-DNA binding.
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PMID:A novel point mutation in the 3' flanking region of the DNA-binding domain of topoisomerase II alpha associated with acquired resistance to topoisomerase II active agents. 754 41

Heparan sulfate proteoglycans are thought to be obligatory for receptor binding and subsequent mitogenic activity of basic fibroblast growth factor (FGF-2). In a previous study (Nurcombe V., Ford, M. D., Wildschut, J., Bartlett, P. F. (1993) Science 260, 103-106) we have shown that primary cultures of mouse neuroepithelial cells and a cell line derived from then, 2.3D, secrete a heparan sulfate proteoglycan with a high affinity for FGF-2. In this study, a combination of affinity chromatography and gel chromatography was used to further isolate heparan sulfate side chains with high affinity for FGF-2. These active chains had an average molecular weight of 18,000-20,000. In order to determine whether heparan sulfate chains with specificity for FGF-2 also displayed selectivity for the different FGF receptors, peptides designed to the heparin-binding region of the receptors were used in competitive inhibition studies. The structure of the predicted heparin-binding domain of the FGF receptor 1 was modeled on the basis of its presumed secondary and tertiary structure homology with immunoglobulin loops. These results suggested that many of the basic residues within the second immunoglobulin loop of the FGF receptor 1 form a basic domain in the molecule and therefore form part of a heparin-binding site. Peptides homologous to this region of FGF receptor 1 were shown to inhibit mitogenesis in 2.3D cells, while those to FGF receptor types 2, 3, and 4 did not. A reverse transcriptase-polymerase chain reaction assay designed to detect expression of the four FGF receptors types demonstrated that FGF receptors 1 and 3 were present on the 2.3D cell line but that receptors 2 and 4 were not. These findings indicate that unique heparan sulfate domains interact with specific cell-surface receptors to direct cellular responses.
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PMID:Heparan sulfates mediate the binding of basic fibroblast growth factor to a specific receptor on neural precursor cells. 755 20

By reverse transcriptase polymerase chain reaction on messenger RNA from human polymorphonuclear cells, we have isolated a sequence identical to the cDNA coding for intracellular interleukin 1 receptor antagonist (icIL-1ra), but containing an additional in-frame 63-bp sequence located three codons downstream of the translation start of icIL-1ra. This additional sequence is inserted between the first and second exon of the intracellular form, the latter of which is colinear with part of the first exon of the secreted form of IL-1ra. The additional sequence is coded by an extra exon located 2 kb downstream the first icIL-1ra-specific exon. The complementary DNA sequence of the alternatively spliced form of icIL-1ra shows that the predicted protein differs from classical icIL-1ra in the NH2 terminus by insertion of a leaderless sequence of 21 amino acids rich in glycine and glutamic acid residues. Transcripts coding for this new form of icIL-1ra were detected in activated fibroblasts, keratinocytes, and at low levels in myelomonocytic cells. The recombinant protein expressed in COS cells had an apparent molecular mass in sodium dodecyl sulfate polyacrylamide gel electrophoresis of 25 kD compared to 22 kD of classical icIL-1ra, and was mostly intracellular. The ability of this new form of icIL-1ra to inhibit IL-1 activity, in terms of induction of E-selectin and human immunodeficiency virus replication, was comparable to that of classical icIL-1ra. We propose to refer to this new form of icIL-1ra as icIL-1ra type II.
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PMID:Cloning and characterization of a new isoform of the interleukin 1 receptor antagonist. 762 20


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