RNA-directed DNA Polymerase Activity Research Articles

Early DNA-RNA Complex from the Endogenous RNA-directed DNA Polymerase Activity of Uninfected Chicken Embryos

WHEN endogenous DNA polymerase activity was isolated from normal uninfected chicken embryos1, we suggested that it was RNA-directed for several reasons. The activity was sensitive to RNAase, but was completely resistant to DNAase and partially resistant to actinomycin D; and the DNA product of the chicken endogenous DNA polymerase activity hybridized to RNA isolated from the same fraction from chicken embryos. Both the template and the DNA polymerase of this chicken endogenous DNA polymerase activity were unrelated to the RNAs and DNA polymerases of Rous sarcoma or reticulo-endotheliosis viruses. Further characterization of the RNA tumour virus endogenous DNA polymerase activity as RNA-directed came from the isolation of a complex of RNA and early DNA product2–5. This complex sedimented rapidly in sucrose gradients and banded at the density of RNA and RNA-DNA hybrids in Cs2SO4 equilibrium density gradients. The complex was disrupted by RNAase (which breaks down RNA), alkali (which breaks down RNA and denatures DNA) and heat (which denatures DNA and RNA-DNA hybrids, but leaves intact covalently bound RNA-DNA). However, previously we were not able to isolate such a complex from the chicken endogenous DNA polymerase activity.

Endogenous RNA-directed DNA polymerase activity in uninfected chicken embryos

Endogenous RNA-directed DNA polymerase activity in uninfected chicken embryos

A single subunit from avian myeloblastosis virus with both RNA-directed DNA polymerase and ribonuclease H activity.

Two structurally distinct forms of RNA-directed DNA polymerase from avian myeloblastosis virus were resolved by chromatography on phosphocellulose and purified. In addition to RNA-directed DNA polymerase activity, both enzymes had ribonuclease H (RNase H) activity, which degraded the RNA moiety of RNA.DNA hybrids. As determined by sodium dodecyl sulfate-polyacrylamide disc-gel electrophoresis, one form had two subunits, alpha (alpha) and beta (beta), with molecular weights of 65,000 and 105,000, respectively. The other had a single subunit, alpha, with a molecular weight of 65,000. The sedimentation coefficients of alphabeta and alpha, determined by glycerol gradient centrifugation in 0.35 M KCl, were 7.8 S and 5.2 S, respectively. Both enzymes had similar antigenic determinants and could not be distinguished by a differential response to several different RNA and DNA templates. We suggest that alpha, which contains both RNA-directed DNA polymerase and RNase H activity, is derived by dissociation of alphabeta; the function of the beta subunit is unknown.

Karyotypic, virologic, and immunologic analyses of two continuous lymphocyte lines established from New Zealand black mice: possible relationship of chromosomal mosaicism to autoimmunity.

Two continuous-suspension lymphocyte lines were isolated from the spleen and fibrosarcoma of a New Zealand Black female mouse. A C-type virus with a density of 1.16 g x cm(-3), 70S RNA, and RNA-directed DNA polymerase activity was isolated. The virus was infectious for NRK, NZB, (NZB x NZW)F(1), and (NZW x NZW)F(1) embryos, and for BALB/c 3T3 cells, but not for NIH Swiss cells. All cells from established lymphocyte cultures, as well as some embryo cells from New Zealand Black mice, showed karyotypic abnormalities. The possibility of chromosomal mosaicism is suggested.

Endogenous RNA-directed DNA polymerase activity in uninfected chicken embryos.

Early chicken embryos that are either positive or negative for group-specific antigens of avian leukosis viruses contained endogenous RNA-directed DNA polymerase activity. This endogenous DNA polymerase activity was not increased after mixture of soluble DNA polymerases isolated from chicken embryos with disrupted chicken embryo cells. The endogenous activity was resistant to treatment with deoxyribonuclease, and the initial rate of DNA synthesis was partially resistant to actinomycin D. In contrast, over 90% of the endogenous polymerase activity was destroyed by ribonuclease in medium with high salt concentration. The DNA product of the endogenous DNA polymerase activity from chicken embryos did not hybridize with RNA of Rous sarcoma virus or reticuloendotheliosis virus, whereas about 40% of this DNA product hybridized with the RNA from the same chicken-cell fraction. Antibody against DNA polymerase of avian myeloblastosis virus did not neutralize the chicken endogenous DNA polymerase activity. These results demonstrate that uninfected chicken embryo cells contain endogenous RNA-directed DNA polymerase activity that is not derived from avian leukosis or reticuloendotheliosis viruses.

3-Cyclic amine derivatives of rifamycin: strong inhibitors of the DNA polymerase activity of RNA tumor viruses.

Derivatives of rifamycin-SV with substituted cyclic-amine side chains in position 3 of the ansa ring are strong inhibitors of RNA-directed DNA and DNA-directed DNA polymerase activity of RNA tumor viruses of murine, feline, and avian origin. Among 37 3-amine derivatives of rifamycin-SV that were tested, 29 3-cyclic amine derivatives were good inhibitors of the viral polymerases. Especially active were 3-piperidyl derivatives of rifamycin-SV with cyclohexyl and cyclohexylalkyl substituents. Derivatives that were effective against the viral polymerase also blocked cell transformation by the murine sarcoma virus. A DNA-directed DNA polymerase preparation from human KB cells was less sensitive to inhibition by these derivatives than the virion polymerase.

Preparation of RNA-directed DNA polymerase from spleens of [formula omitted] mice infected with Rauscher leukemia virus

A high level of RNA-directed DNA polymerase activity is found in spleens of Balb c mice infected with Rauscher murine leukemia virus. The enzyme is associated with sedimentable subcellular fractions, can be separated from other cellular DNA polymerases, and is present in useful quantity for purification purposes. Complete solubilization of the enzyme, achieved and maintained by using a combination of detergent and high salt solution, is required for a satisfactory purification both in the initial extraction procedure and in subsequent column chromatography.

RNA-Directed DNA Synthesis and RNA Tumor Viruses

Publisher Summary The discovery of RNA-directed DNA synthesis in disrupted virions of RNA tumor viruses added strong support to the hypothesis that information transfer from RNA to DNA exists in biological system. The chapter discusses the properties of the endogenous reaction carried out by the virion DNA polymerase. To study the endogenous reaction disrupted, virions are incubated with substrates in the absence of any added template and synthesis of DNA is observed using the RNA present in the virions as template. The chapter also discusses the general implications of RNA-directed DNA synthesis in relation to tumor viruses, neoplastic cells, and normal cells. It is hypothesized that the leukovirus RNA-directed DNA polymerase activity is an integral part of the ribonucleoprotein core of the virions. The cores are synthesized in cells as precursor particles and then are incorporated into complete virions when the virions are assembled by budding at the cell surface. This core enzyme system contains not only the template-primer RNA, a DNA polymerase that can transfer information from RNA to double-stranded DNA, but ancillary enzymes, such as polynucleotide ligase and nucleases, which may aid in integrating the viral information with cellular DNA. This suggests that the virion enzyme activity is related to normal cellular DNA polymerases, and that there are homologies between the amino acid sequences of the viral enzyme and normal cellular enzymes. The relationship of RNA-directed DNA synthesis to neoplasia depends upon the relationship of RNA tumor viruses to neoplasia, which is supported by three general hypotheses: the provirus model, the oncogene model, and the protovirus model.