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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)

We have investigated three aspects of RNA turmor virus replication and cell transformation: (1) the properties of the purified avian and mammalian viral RNA-directed DNA polumerase, (2) some characteristics of the viral 60-70S RNA genome, 30-40S RNA subunits and intracellular viral RNA species, and (3) the interaction of the viral DNA polymerase with its RNA template early during infection and cell transformation by the murine sarcoma-leukemia virus (MSV[MLV]). Avian myeloblastosis virus (AMV) contains two forms of RNA-directed DNA polymerase, alpha, consisting of a single polypeptide of molecular weight 65,000, and alphabeta, consisting of two polypeptides of molecular weights 65,000 and 105,000. The alpha and alphabeta forms of AMV DNA polymerase both possess RNase H activity that requires free end termini on the ribopolymer and can degrade the RNA of the RNA-DNA hybrid in the 3' to 5' and 5' to 3' directions. But, alpha and alphabeta possess a different mode of exoribonuclease activity. While alphabeta RNase H is a processive exoribonuclease that degrades the polynucleotide chain to a core residue before attacking a second chain, alpha RNase H is a random exoribonuclease that releases the polynucleotide after each scission. Highly purified Moloney-MSV(MLV) DNA polymerase has both RNase H activity and the ability to read viral 60-70S RNA. These activities comigrate through five different steps of purification and are present at levels comparable to those found in purified AMV DNA polymerase. The MSV(MLV) 60-70S RNA genome and 35S RNA subunits were shown by periodate oxidationtritiated borohydride reduction to contain adenosine as the major 3'-terminal nucleoside. Poly (A) segments were isolated from viral 60-70S and 35S RNA by treatment with RNase A or RNase T1 and purified by afinity chromatography and gel electrophoresis. Viral poly(A) was shown to be present at the 3' terminus as -G(C,U)A190AOH. The similar sequence reported for poly(A) present in mammalian mRNA suggests that similar mechanisma are involved in the transcription and processing of both cellular and viral DNA sequences. Within transformed cells replicating MSV(MLV), viral 35S and 20S RNA were found in membrane-bound polyribosomes, whereas only 35S RNA was detected in free polyribosomes. The origin and function of 20S RNA is unknown. The early events during rapid infection and cell transformation of mouse 3T6 cells by the Harvey strain of MSV(MLV) were studied. By both autoradiographic analysis and molecular hybridization, viral DNA synthesis was detected in the cytoplasm by 1 hour after infection, reached a maximum at 2 hours, and subsequently decreased. Cytological chase experiments produced evidence that cytoplasmic viral DNA was transported to the nucleus. In situ hybridization experiments using radioactive viral DNA product as a probe demonstrated the rapid association of viral DNA sequences with the chromocenters of interphase nuclei and with the centromeric heterochromatin regions of some chromosomes.
Cold Spring Harb Symp Quant Biol 1975
PMID:Properties of oncornavirus RNA-directed DNA polymerase, the RNA template, and the intracellular products formed early during infection and cell transformation. 5 Sep 2

We developed a non-radioisotopic (non-RI) reverse transcriptase assay (RTA). The reverse transcriptase (RT) incorporates biotin-11-deoxyuridine-triphosphate (bio-dUTP) using a poly(rA) template hybridized with oligo(dT) primer that is immobilized on the surface of a 96-well microtiter plate. This assay is thus semi-automated by adapting it to an ELISA testing format. The incorporation of bio-dUTP was enhanced by adding cold dTTP to the reaction mixture, optimally in a molar ratio 4:1 (dTTP:bio-dUTP). This non-RI RTA is more sensitive than the conventional RI assay for the detection of purified Rous-associated virus 2 (RAV-2) and of human immunodeficiency virus type 1 (HIV-1) lysate. Because of its simple procedure, higher sensitivity and non-use of RI materials, the assay can be utilized not only for virological studies but also for routine safety screening of biological products for retroviral contamination.
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PMID:A non-radioisotopic reverse transcriptase assay using biotin-11-deoxyuridinetriphosphate on primer-immobilized microtiter plates. 128 Jun 40

The hormone gastrin is mainly produced by the G cells of the antral mucosa and plays a major role in the regulation of digestive mucosal growth. Since it permits identification of cell types containing mRNA, in situ hybridization (ISH) appears to be an interesting method for studying gastrin-producing tissues. In this study, in situ detection of gastrin mRNA has been carried out on frozen sections of four human normal antral mucosa samples and of six colonic carcinomas removed from patients with high levels of plasma gastrin, using a gastrin oligonucleotidic DNA probe. We have compared the results provided respectively by the [35s] labelling and the digoxigenin labelling of the synthetic probe. Positive cells were found in each normal sample analysed with radioactive- as well as digoxigenin-labelled antisense probes. The total number of cells expressing gastrin mRNA appeared slightly higher with the [35s]-labelled probe, while the digoxigenin-labelled probe gave a better definition of positive signals. In contrast, neither radioactive nor cold probes gave positive signals in the six colonic carcinoma samples, although gastrin expression had been demonstrated in these tumours using a reverse transcriptase-PCR method. These results show that, although ISH does not seem sensitive enough to allow the detection of very low levels of gastrin expression, it would appear to be a reliable method for visualizing gastrin mRNA in human antral mucosa.
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PMID:Detection of gastrin mRNA by in situ hybridization using radioactive- and digoxigenin-labelled probes: a comparative study. 144 1

We studied the inactivation of the etiologic agent of the acquired immunodeficiency syndrome, human immunodeficiency virus, in the course of the Kistler and Nitschmann cold ethanol fractionation of human blood plasma. By measuring reverse transcriptase activity and viral infectivity, we have shown that the virus load is reduced by a factor of 10(4) during the initial and at least a factor of 10(6) during the subsequent steps of the fractionation procedure. This loss of virus may be observed in the absence or in the presence of antibodies against human immunodeficiency virus and is due to a combination of chemical inactivation, physical partition, and injury caused by repeated freezing and thawing. The laboratory data therefore further confirm epidemiological studies which indicate that immunoglobulin preparations obtained by ethanol fractionation do not transmit human immunodeficiency virus.
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PMID:Inactivation and partition of human immunodeficiency virus during Kistler and Nitschmann fractionation of human blood plasma. 245 74

The gap between early molecular evolution and the origin of the first cell may have been bridged by a photoheterotrophic obcell, consisting of genes and ribosomes attached to the outer surface of a phospholipid vesicle containing a light-driven proton pump and a proton-driven pyrophosphate synthase. I argue that the obcell was the substratum for the origin of DNA replication; DNA segregation by the growth and division of the peptidoglycan murein; periplasmic solute-binding proteins; bioenergetics, including the F0F1 proton-driven ATP synthase; active transport of calcium; and facilitated diffusion of nutrients across membranes, and that it played the major role in the replacement of ribozymes by protein catalysts. Curved growth of the peptidoglycan and a mutation causing septum formation produced the first true cell. Evolution of porins, sodium extrusion and potassium import, conversion of the facilitated diffusion proteins to active pumps, and the evolution of intermediary metabolism, carbon and nitrogen fixation, and of substrate level phosphorylation, completed the origin of the first negibacterial eubacterium, from which all other cells evolved, and from which they have inherited most of their major catalytic properties--with the notable exceptions of reverse transcriptase, RNA splicing, and methanogenesis, all of which I believe evolved very much later.
Cold Spring Harb Symp Quant Biol 1987
PMID:The origin of cells: a symbiosis between genes, catalysts, and membranes. 345 90

Polyguanylic acid was found to be a potent inhibitor of RNase H associated with mammalian viral reverse transcriptase, indicating a strong interaction between polyguanylic acid and the reverse transcriptase protein. Based on this observation, we have developed three simple procedures for the purification of mammalian viral reverse transcriptases. In the first procedure, a nucleic acid-free extract of Rauscher murine leukemia virus was applied to a column of phosphocellulose and the reverse transcriptase was eluted by a low concentration (50 microM) of polyguanylic acid. Polyadenylic acid and polyuridylic acid could not replace polyguanylic acid for the elution. In the second procedure, a polyuridylic acid-Sepharose column was substituted for phosphocellulose, and the elution was again achieved by polyguanylic acid. In the third affinity procedure, the reverse transcriptase in a nucleic acid-free viral extract was incubated in the cold with 50 microM polyguanylic acid and the complex was adsorbed onto a DEAE-cellulose column. After washing to remove uncomplexed and weakly complexed proteins, the reverse transcriptase was eluted in a concentrated form at 0.3 M NaCl with a recovery of greater than 70%. by polyacrylamide gel analysis in the presence of sodium dodecyl sulfate, the enzyme appeared to be nearly pure.
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PMID:Simple affinity procedure for the purification of mammalian viral reverse transcriptases. 616 Feb 61

Two DNA polymerases with properties of viral RNA-directed DNA polymerase were found in the placenta of a patient with breast cancer. Both enzyme activities were purified by column-chromatographic procedures or by preparative isoelectric focusing. The most distinguishing feature of the two enzymes is their specificity to transcribe (rA)n . (dT)12 or (rC)n . (dG)18. The two enzymes differ with respect to their elution profiles from the phosphocellulose column, isoelectric point, molecular weight, bivalent-cation requirements and thermal stability. Serological analysis of the (rA)n . (dT)12-activated enzyme showed that this enzyme is immunologically not related to DNA polymerase-gamma, or to any of the reverse transcriptases purified from retroviruses of avian, murine and subprimate origin. However, the activity of this enzyme was neutralized by antibodies to reverse transcriptase purified from human spleen of a patient with myelofibrosis [Chandra & Steel (1977) Biochem. J. 167, 513-524]. Attempts to purify reverse transcriptase of normal human placenta were repeatedly unsuccessful. Once the crude homogenate of normal placenta was freed from endogenous nucleic acids, no (rC)n . (dG)18-dependent activity cold be detected.U
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PMID:Evidence for two forms of reverse transcriptase in human placenta of a patient with breast cancer. Purification and biochemical characterization of the enzymes. 617 35

RNA isolated by conventional guanidinium isothiocyanate methods from tissues of a mollusc (red abalone: Haliotis rufescens) is largely degraded and discolored by contaminants. These contaminants are associated with inhibition of reverse transcriptase, prevent accurate spectrophotometric determination of RNA concentration, and impart undesirable viscosity to the preparations. A cold two-step method of RNA isolation was devised which provides high yields of full-length RNA templates from these tissues and eliminates the discolored contaminant. Immediately following homogenization of tissues at ca. 5 degrees C, which proved crucial for the recovery of high-molecular weight species, the RNA is isolated from the bulk of the RNase by a single acid-phenol-chloroform extraction at 0 degrees C. The inhibitor of reverse transcriptase, suspected to be a proteoglycan (or a similar high-molecular-weight polyanion) component of the intestinal mucus, is eliminated only by a second purification step employing ultracentrifugation through a dense cushion of CsCl. This cold two-step method should prove useful for providing full-length RNA templates relatively free of polysaccharide, a common contaminant of RNA preparations, from both plant and animal tissues.
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PMID:Isolation of full-length RNA templates for reverse transcription from tissues rich in RNase and proteoglycans. 768 67

Highly purified progenitors (including erythroid [BFU-E], granulo-monocytic [CFU-GM], multipotent [CFU-GEMM] progenitors, as well as multipotent progenitors with self-renewal capacity [CFU-B]) express high-affinity growth factor receptors (GFRs), with prevalent interleukin-3 receptors (IL-3Rs) (2,700/cell), a > or = 10-fold lower number of IL-6Rs (145/cell) and granulocyte-macrophage colony-stimulating factor receptors (GM-CSFRs) (300/cell), and a barely detectable level of erythropoietin (Ep) receptors (75/cell). Hematopoietic growth factor (HGF) dosages inducing peak clonogenetic effects are associated with partial/subtotal occupancy of the homologous HGF receptor (HGFR). Cross-reactivity between GFRs and heterologous GFs (including IL-6, IL-3, GM-CSF, Ep, and the kit ligand [KL]) was explored by competition experiments on purified progenitors with radiolabeled and excess cold HGFs at +4 degrees C. No cross-reaction was observed between IL-6R, IL-3R, EpR, and the heterologous GFs, whereas the GM-CSFR showed cross-reactivity with IL-3 and, to a lesser extent, KL. Modulation of GFRs was examined after 18 or 40 hours of incubation with GF(s) at 37 degrees C, followed by ligand-binding assay at 20 degrees C. IL-6, IL-3, GM-CSF, and Ep induce a marked down-modulation of their own receptors. Interestingly, each GF induces the transactivation of the R(s) for the "distal" GF(s): (1) IL-6 induces transactivation of IL-3R, but not of GM-CSFR/EpR; (2) IL-3 causes a rapid upmodulation of GM-CSFR/EpR ("pure" progenitors treated with IL-3 show upmodulation of GM-CSFR alpha-chain mRNA by reverse transcriptase-polymerase chain reaction); whereas (3) GM-CSF induces the transactivation of the EpR. This chain upmodulation of HGFRs may underlie the synergistic interactions between the HGFs in clonogenetic culture. It is emphasized that KL does not induce upmodulation of the other GFRs. Finally, Ep, GM-CSF, and IL-3 do not modulate the expression of the "proximal" HGFRs (ie, GM-CSFR/IL-3R/IL-6R, IL-3R/IL-6R, and IL-6R, respectively). These results allow insight into the cellular basis of hematopoiesis, ie, the complex and coordinate interactions between HGFs and their receptors. They are compatible with a model of cascade transactivation via upmodulation of GFRs in the initial key steps of hematopoietic differentiation, whereby the action of each GF enhances the effect of the distal GF(s) by a multistep chain-potentiation mechanism.
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PMID:Cascade transactivation of growth factor receptors in early human hematopoiesis. 845 93

In a recent study, antioxidant therapy at the time of renal transplantation in humans was associated with fewer rejection episodes and extended graft survival. A hypothesis generated by such studies and based on the response-to-injury model is that reducing the oxidative injury during transplantation may dampen certain cellular responses to injury that are important in triggering allograft rejection. To test whether ablation of oxidative injury would limit such responses, kidneys were transplanted between Wistar-Furth rats, with and without antioxidant 21-aminosteroid. 21-Aminosteroid was administered before kidney harvest and, again, before transplant reperfusion. The recipient's left kidneys, removed to accommodate the donor kidneys, were used as normal control. The removal of the right kidneys contralateral to the transplant were delayed to day 4 to provide interim renal support. The transplanted kidneys were harvested on day 7. Administration of 21-aminosteroid was associated with better graft function and reduced lipid peroxidation. Compared with the normal control kidneys, the kidneys transplanted with vehicle had higher cytokine mRNA levels (measured by reverse transcriptase-polymerase chain reaction) for interleukin 2, interleukin 6, tumor necrosis factor-alpha, and interferon-gamma. The levels for these cytokines were reduced in kidneys transplanted with 21-aminosteroid. An increase in inducible nitric oxide synthase mRNA in the transplanted kidney was inhibited by 21-aminosteroid, as were the increase in class I and II MHC antigens. The new finding, that a reduction in transplantation-related oxidative injury in a syngeneic model is accompanied by a reduction in the expression of cytokines, MHC antigens, and inducible nitric oxide synthase, provides partial support for the response-to-injury hypothesis in the setting of renal transplantation. The data also demonstrate for the first time the efficacy of 21-aminosteroid to reduce lipid peroxidation and renal injury in kidneys transplanted after cold preservation.
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PMID:Antioxidant lazaroid U-74006F improves renal function and reduces the expression of cytokines, inducible nitric oxide synthase, and MHC antigens in a syngeneic renal transplant model. Partial support for the response-to-injury hypothesis. 897 Jun 19


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