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- W2025247138 abstract "We have compared the relative merits of several procedures for the isolation of RNA-directed DNA polymerase (EC 2.7.7.7) from cells using a reconstituted model system consisting of a mixture of woolly monkey (simian) sarcoma virus and a cultured human lymphoblastoid cell line, NC-37. When the cell-virus mixture was gently disrupted and fractionated by differential centrifugation, most of the added polymerase was recovered associated with a particulate fraction obtained from the post-mitochondrial supernatant. Purification of the polymerase was best achieved starting from this fraction. The particulate fraction itself can be purified by gel filtration through a Sepharose 2 B column. This procedure did not significantly alter the composition of viral and cellular DNA polymerases. Wheras as little as 7.5 · 105 viral particles were sufficient for the detection of RNA-directed DNA polymerase activity, a minimum of about 1011 particles were necessary for the isolation and unequivocal characterization of the enzyme from the cell-virus mixture by subcellular fractionation and chromatographic separation from cellular DNA polymerases. Purified RNA-directed DNA polymerase had the same primer-template characteristics, sedimentation properties, and immunological cross reactivity as the enzyme purified from density gradient-banded virions of simian sarcoma virus. Methods involving total extraction of the cell-virus mixture either by repeated freezing and thawing followed by detergent treatment or by Dounce homogenization and treatment with high salt and detergent failed to provide RNA-directed DNA polymerase free of cellular DNA polymerases. Because of this, low levels of cellular RNA-directed DNA polymerase may be missed when these approaches are used." @default.
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- W2025247138 title "A comparative evaluation of methods for isolation of RNA-directed DNA polymerase from cells in a reconstituted system" @default.
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- W2025247138 doi "https://doi.org/10.1016/0005-2787(76)90190-8" @default.
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