TY - JOUR
T1 - Covalently closed circular DNA of avian sarcoma virus
T2 - Purification from nuclei of infected quail tumor cells and measurement by electron microscopy and gel electrophoresis
AU - Guntaka, Ramareddy V.
AU - Richards, Oliver C.
AU - Shank, Peter R.
AU - Kung, Hsing Jien
AU - Davidson, Norman
AU - Pritsch, Edward
AU - Bishop, J. Michael
AU - Varmus, Harold E.
N1 - Funding Information:
We thank S. Heasley, L. Fanshier, and J. Jackson for expert technical assistance, and B. Cook for typing the manuscript. This work w&s supported by American Cancer Society (VC-70), U.S. Public Health Services grant (12705), and Contract nos NO1 CP 33293 and NO1 CP 43306 within the Virus Cancer Program of the National Cancer Institute, National Institutes of Health, Public Health Services. One of us (0. C. R.) was on sabbatical leave under the terms of a NIH Research Fellowship award (CA01985). Another author (H. E. V.) is a recipient of a Research Careor Development award (CA70193) from the National Cancer Institute. Another author (P. R. S.) acknowledges support from Public Health Service Training grant 5TOl CA 05303 and a fourth author (R. V. G.) acknowledges support from the California Division of the American Cancer Society (D-235).
PY - 1976/9/15
Y1 - 1976/9/15
N2 - Avian sarcoma virus-specific DNA is synthesized three to fourfold more efficiently after infection of a Japanese quail tumor cell line (QT-6, derived from a fibrosarcoma induced with methylcholanthrene) than after infection of duck or quail embryo fibroblasts; 10 to 30 copies of viral DNA per cell are generally observed in the first 24 hours after infection at multiplicities of one to five focus-forming units per cell. During the first five hours after infection viral DNA accumulates only in the cytoplasm of QT-6 cells; thereafter viral DNA is also observed in the nucleus. Covalently closed circular forms of viral DNA (form I) accumulate exclusively in the nucleus, and they persist for at least 600 hours after infection; within 24 to 48 hours after infection, form I DNA constitutes as much as 50% of the nuclear species of viral DNA and 20 to 25% of viral DNA in whole cells. We have purified viral form I DNA about 105-fold from acutely-infected QT-6 cells by cell fractionation, sodium dodecyl sulfate/sodium chloride precipitation of cellular DNA, equilibrium density centrifugation in cesium chloride gradients containing propidium diiodide, rate zonal sedimentation in sucrose gradients, and chromatographic selection of molecules renaturing with zero-order kinetics. The length of viral form I DNA has been measured by direct observation in the electron microscope, revealing species of 6.6 × 106, 5.6 × 106 and 2 to 4 × l06 daltons. These species presumably correspond to DNA transcribed from RNA subunits of avian sarcoma virus (3.3 × 106 daltons); DNA transcribed from RNA subunits of transformation-defective deletion mutants of the virus (2.8 × 106 daltons) shown to be present in the infecting stock; and newly identified, incomplete transcripts of these RNA subunits. The size of these species and their viral specificity were confirmed by molecular hybridization after electrophoresis in agarose gels. Viral form I DNA of 5 to 7 × 106 daltons is infectious in chick embryo cells, resulting in production of avian sarcoma virus and transformation-defective virus, whereas viral form I DNA of 2 to 4 × 106 daltons is not infectious in similar tests and is presumably defective.
AB - Avian sarcoma virus-specific DNA is synthesized three to fourfold more efficiently after infection of a Japanese quail tumor cell line (QT-6, derived from a fibrosarcoma induced with methylcholanthrene) than after infection of duck or quail embryo fibroblasts; 10 to 30 copies of viral DNA per cell are generally observed in the first 24 hours after infection at multiplicities of one to five focus-forming units per cell. During the first five hours after infection viral DNA accumulates only in the cytoplasm of QT-6 cells; thereafter viral DNA is also observed in the nucleus. Covalently closed circular forms of viral DNA (form I) accumulate exclusively in the nucleus, and they persist for at least 600 hours after infection; within 24 to 48 hours after infection, form I DNA constitutes as much as 50% of the nuclear species of viral DNA and 20 to 25% of viral DNA in whole cells. We have purified viral form I DNA about 105-fold from acutely-infected QT-6 cells by cell fractionation, sodium dodecyl sulfate/sodium chloride precipitation of cellular DNA, equilibrium density centrifugation in cesium chloride gradients containing propidium diiodide, rate zonal sedimentation in sucrose gradients, and chromatographic selection of molecules renaturing with zero-order kinetics. The length of viral form I DNA has been measured by direct observation in the electron microscope, revealing species of 6.6 × 106, 5.6 × 106 and 2 to 4 × l06 daltons. These species presumably correspond to DNA transcribed from RNA subunits of avian sarcoma virus (3.3 × 106 daltons); DNA transcribed from RNA subunits of transformation-defective deletion mutants of the virus (2.8 × 106 daltons) shown to be present in the infecting stock; and newly identified, incomplete transcripts of these RNA subunits. The size of these species and their viral specificity were confirmed by molecular hybridization after electrophoresis in agarose gels. Viral form I DNA of 5 to 7 × 106 daltons is infectious in chick embryo cells, resulting in production of avian sarcoma virus and transformation-defective virus, whereas viral form I DNA of 2 to 4 × 106 daltons is not infectious in similar tests and is presumably defective.
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U2 - 10.1016/0022-2836(76)90090-5
DO - 10.1016/0022-2836(76)90090-5
M3 - Article
C2 - 185393
AN - SCOPUS:0017186696
SN - 0022-2836
VL - 106
SP - 337
EP - 357
JO - Journal of Molecular Biology
JF - Journal of Molecular Biology
IS - 2
ER -