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)

We reported earlier that core preparations of Rauscher murine leukemia virus, when separated on an isopycnic sucrose gradient, did not contain detectable levels of RNase H activity, while retaining high levels of reverse transcriptase activity. We reexamined this phenomenon, and the earlier observation was found to be reproducible. However, when doubly banded preparations of viral cores were solubilized and reverse transcriptase was isolated by ion-exchange chromatography, a coincident peak of a nuclease activity with the specificity of RNase H was observed, which indicated that RNase H was selectively inhibited in the core fractions. By direct activity measurements using the purified reverse transcriptase-RNase H from cores, this endogenous inhibitor has been identified as the viral RNA. Viral 70S RNA strongly inhibited RNase H activity purified either from whole virions or from prefractionated cores. Other RNAs tested that had inhibitory effects were yeast tRNA, polyadenylic acid, and polyguanylic acid. Polyuridylic acid and polyadenylic acid were moderately inhibitory, and polycytidylic acid did not inhibit the RNase H. A rabbit anti-reverse transcriptase immunoglobulin G inhibited both the reverse transcriptase and RNase H activities of the enzyme purified from cores. These data provide a rational explanation for the failure to detect RNase H activity in core preparations of Rauscher murine leukemia virus. Furthermore, these data are consistent with the idea that the RNase H and reverse transcriptase activities purified from cores reside on the same protein molecule. Possible biological implications of the observed inhibition of RNase H by RNA is discussed.
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PMID:Inhibition by RNA of RNase H activity associated with reverse transcriptase in Rauscher murine leukemia virus cores. 8 12

RNase H of a temperature-sensitive mutant of Rauscher murine leukemia virus is thermolabile, establishing this activity as a virus-coded function of the mammalian type C virus reverse transcriptase.
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PMID:Mammalian retrovirus-associated RNase H is virus coded. 8 14

An RNA-direct DNA polymerase was purified from human melanoma tissue by successive column chromatography on DEAE-cellulose (DE-23 and DE-52) and phosphocellulose. The purified reverse transcriptase has a mol. wt. of 68,000, a pH optimum of 8.0, a Mn2+ optimum of 0.6 mM, and a KCl optimum of 60 mM. The purified enzyme transcribes (rA)n - (dT)12, (rC)n - (dG)18, (Ome-rC)n - (dG)18 and a 70s RNA from Rauscher leukemia virus (RLV), but failed to transcribe (dA)n - (dT)12. This enzyme has no terminal deoxynucleotidyl transferase activity. Serological studies have shown that the reverse transcriptase from human melanoma tissue is antigenically not related to DNA polymerases from Simian sarcoma virus (SiSV), Avian myeloblastosis virus (AMV), RLV, and human spleen of a patient with myelofibrosis. The purified enzyme showed a close antigenic resemblance to DNA polymerases from baboon endogenous virus (BEV) and rhabdomyosarcoma virus (RD-114), the endogenous virus of the cat.
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PMID:Biochemical and immunological characterization of a reverse transcriptase from human melanoma tissue. 8 88

A retrovirus antigenically distinct from known type C, B and D viruses was isolated from normal mink (Mustela vison) lung cells that had been co-cultivated with 5-iododeoxyuridine- and dexamethasone-treated dog mammary tumour cells. Cytogenetic studies of the virus-releasing co-culture showed mitotic figures identical to the normal mink cell line (MvlLu) with the exception of a low frequency of cells with extensive chromosomal breakage and uncoiling. The new virus bands at a buoyant density of 1.16 g/ml, contains 60S RNA and a reverse transcriptase which prefers Mn2+ over Mg2+ for the synthesis of DNA. This enzyme utilizes poly(rA).oligo(dT) more efficiently than poly(dA).oligo(dT) and is also able to synthesize DNA copies from the endogenous RNA. Morphologically, it is a typical type C virus. Filtered virus readily infects mink, dog and other mammalian cells indicating the amphotropic nature of its cell growth requirement. Hybridization studies showed that normal mink DNA contains multiple copies of proviral sequences of this newly isolated virus. Serological analyses indicate that the mink endogenous virus contains in its core protein, in addition to the interspecies type-C determinant, an antigenic component related to one of the determinants found in the feline leukaemia virus p30 protein. This determinant is not present in the Rauscher leukaemia virus, RD114 virus or simian sarcoma virus.
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PMID:Characterization of a retrovirus isolated from normal mink cells co-cultivated with a dog mammary tumour. 8 51

Permanent cell lines have been established from twelve diffuse histiocytic lymphomas (SU-DHL-1 to -12), three American Burkitt's lymphomas (SU-AmB-1 to -3), two acute lymphoblastic leukemias (SU-ALL-1 and -2), and three diffuse undifferentiated lymphomas (SU-DUL-1, -2, and -3). The cultured cells displayed neoplastic characteristics, as manifested by heterotransplantability in congenitally athymic nude mice and by the presence of cytogenetic abnormalities in early passage generations. Functional and marker studies revealed that the three American Burkitt's lymphomas, as well as several of the diffuse histiocytic and undifferentiated lymphomas, were of B-lymphocytic origin, whereas the two acute lymphoblastic leukemias were both of T-lymphocytic origin. Two of the cell lines, SU-DHL-1 and -2, appeared to be of true histiocytic origin; two others exhibited no markers and were designated as "null" cells. All ten of the DHL cell lines studied to date, as well as SU-DUL-1, have been devoid of Epstein-Barr virus (EBV) genomes by the EBNA test, whereas two of the three American Burkitt's lymphoma cell lines were positive. Spontaneous production of a C-type RNA virus was first detected in post-mitochondrial cytoplasmic fractions and culture fluids of the SU-DHL-1 cell line. Screening assays for the detection of reverse transcriptase-positive particles in the culture fluids of the other cell lines indicate that eight of the fifteen cell lines tested to date have spontaneously initiated C-type RNA virus production. After partial purification by ion-exchange and affinity chromatography, the reverse transcriptases of the virus isolated from SU-DHL-1 cells is partially inhibited by antibodies to the reverse transcriptases of C-type viruses of subhuman primate and endogenous feline, but not of murine, origin. Conversely, antibody prepared against the purified SU-DHL-1 viral reverse transcriptase, at concentrations which maximally inhibit the homologous enzyme, partially inhibits the reverse transcriptases of subhuman primate C-type viruses, but has little or no inhibitory activity against the reverse transcriptases of feline or murine leukemia viruses. The viruses produced by the SU-DHL-1 and SU-AmB-3 cell lines have been shown to be infectious for normal human peripheral blood mononuclear cells, normal human bone marrow cells, and certain human lymphoblastoid cell lines. After infection by these viruses, normal human peripheral blood mononuclear cells and human bone marrow cells have exhibited striking changes in growth behavior and morphology which, though not permanently sustained, have many of the features of abortive transformation.
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PMID:Biology and virology of the human malignant lymphomas: 1st Milford D. Schulz Lecture. 8 2

Interferon (150 units/ml) was used to treat SC-1 and AKR-2B cells which were chronically infected with murine leukaemia virus (MuLV). This led to a 100-fold decrease in the amount of infectious virus released into the medium and a 10-fold decrease in the number of virus particles measured by the virion-associated reverse transcriptase assay. However, there was little change in the amount of cell-associated infectious virus, though nearly twice as many cell-associated virions were counted in electron micrographs. With both types of cells, interferon blocked MuLV replication at the post-budding stage, but it did not change the morphology of the particles produced or their content of virion 70S RNA. Infectious virus assembled on the cell membranes of interferon-treated cells was less stable at 37 degrees C than that grown in the absence of interferon. Release of infectious virus from interferon-treated cells was not inhibited by actinomycin D or cycloheximide, though both agents inhibited virus production in controls. These results show that interferon inhibits MuLV replication through effects on virion assembly; these lead both to the formation of non-infectious particles and of fewer virions. Kinetic analysis further shows that interferon affects MuLV assembly rapidly and induction of an antiviral protein may not be required.
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PMID:Effect of interferon on murine leukaemia virus infection. IV. Formation of non-infectious virus in chronically infected cells. 8 89

omicron-Phenanthroline, a zinc chelating agent, is known to inhibit the DNA polymerase activity of cellular DNA-dependent and viral RNA-dependent DNA polymerases. We find that omicron-phenanthroline does not inhibit the reverse transcriptase-associated RNase H activity of retroviruses. Kinetic studies, using DNA template-primers as an inhibitor of RNase H, suggest that zinc does not play any role in template-primer binding by reverse transcriptase. These results also indicate a distinct binding site for the template and triphosphate substrates. Cellular RNase H from calf thymus and RNase H-II from Rauscher leukemia virus are likewise resistant to omicron-phenanthroline inhibition, implying non-involvement of zinc in the nucleic acid hydrolysis by these enzymes.
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PMID:Reverse transcriptase-associated ribonuclease H does not require zinc for catalysis. 8 44

Buffy coats from 31 patients with a diagnosis of leukemia and 16 normal donors were tested for the presence of a viral-like reverse transcriptase. Eighty-five percent of fresh leukemic buffy coats were positive. Also tested were spleens from 16 patients with hematological disorders and 5 spleens from patients without history of hematological malignancy. The 5 normal spleens were negative. Also negative were 4 spleens from patients with Hairy cell leukemia. From the remaining 12 spleens 7 were positive. Reverse transcriptase measurements can be used to distinguish leukemic from normal buffy coats.
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PMID:On the presence of reverse transcriptase in myelo- and lymphoproliferative disorders. 8 54

Samples of three nonmalignant and seven leukemic human cells were examined for DNA polymerase activity that could be identified as RNA tumor virus reverse transcriptase. Experiments on virus-infected model animal cells provided the basis for cell fractionation procedures, and reconstituted systems of known virus, added to human cells, established a threshold of virus detection by enzyme assay at 1 to 10 particles/cell. DNA polymerase activity with some properties similar to a reverse transcriptase was detected in some of the human leukemic cells. However, parallel analyses of nonmalignant cells showed sufficient similarities to raise serious questions about the specificity of the criteria. Reverse transcriptase activity has been reported to be present in white blood cells from a proportion of cases of leukemia; however, it is concluded from the present study that the usual enzymatic criteria using synthetic template primers, which were used in most of the studies reported, are not sufficient to identify a DNA polymerase activity as viral reverse transcriptase.
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PMID:Detection of reverse transcriptase activity in human cells. 8 60

In the endogenous reverse transcriptase reaction, equine infectious anemia virus is able to synthesize complementary DNA (cDNA) of 8,000 nucleotides in high yield. After 2 h in 50 muM dNTP, about 2.8 mug of cDNA per mg of protein is produced, almost 30% of which is long cDNA. The system thus compares favorably with the other two well-characterized endogenous reaction systems, Moloney murine leukemia virus and avian sarcoma virus. Elongation rates of 100 to 150 nucleotides per min have been observed; these rates are comparable to those seen with purified avian myeloblastosis virus reverse transcriptase and significantly higher than those observed in vivo. In the absence of actinomycin D, equine infectious anemia virus does not require high dNTP levels for either optimal incorporation or long cDNA synthesis. The amount of long cDNA synthesized is maximal at 2 h in 50 muM dNTP; neither longer time nor higher dNTP levels (through 1.8 mM) increased this yield. Half-maximum yield in 2 h was achieved at about 15 muM dNTP, which is very similar to the published K(M)'s for isolated avian and murine reverse transcriptases. Total incorporation, on the other hand, continues to rise slowly through 1 mM dNTP; the half-maximum was 30 to 50 muM dNTP. In the presence of 100 mug of actinomycin D per ml, however, higher dNTP levels are required for long cDNA synthesis. We conclude that equine infectious anemia virus is exceptionally well-suited to studies of the physical organization of the retrovirus genome and to investigations of the mechanism of synthesis of the double-standard cDNA endogenous reaction product.
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PMID:Synthesis of long complementary DNA in the endogenous reaction by equine infectious anemia virus. 8 22


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