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Home → Publications → American Chestnut Proceedings → 1982 → Detection of RNA Dependent-RNA Polymerase Activity in Crude Extracts of Virus-like Particles in Endothia Parasitica Strain EP-43

Detection of RNA Dependent-RNA Polymerase Activity in Crude Extracts of Virus-like Particles in Endothia Parasitica Strain EP-43

The RNA dependent-RNA polymerase activity has been detected in crude virus-like particle extracts of 14-day-old shake cultures of EP-43 grown in glucose yeast extract agar. Extracts were made by homogenizing mycelium in 1M sodium acetate (pH 5.0) in a bead-beater. The homogenate was subjected to a low speed spin. The supernatant was made 0.3M NaC1 and PEG 8000 was added at the rate of 10 g/1,000 ml. The precipitate was collected and subjected to one round of differential centrifugation (10,000x g for 30 minutes and 27,000x g for 90 minutes). The RNA polymerase assay mixture contained ATP, GTP, CTP, 3 H-UTP, magnesium ions, EDTA and crude extract, all buffered in TRIS-HC1 to pH 7.9. Incorporation of 3 H-UNP into TCA-insoluble RnA was shown to be actinomycin D insensitive suggesting that polymerase activity was not DNA dependent. Similar experiments done with Endothia parasitica strain 671B also detected incorporation of label into RNA, but to a lesser extent.


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Author(s): Katherine Harper-Morris

Publication: American Chestnut Proceedings - 1982