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Nucleic Acids Research, 1985, Vol. 13, No. 12 4557-4576
© 1985

Articles

Isolation of a fraction from cauliflower mosaic virus-infected protoplasts which is active in the synthesis of (+) and (–) strand viral DNA and reverse transcription of primed RNA templates

C.M. Thomas1,2, R. Hull1, J.A. Bryant2 and A.J. Maule1

1John Innes Institute Colney Lane, Norwich NR4 7UH 2Department of Plant Science, University College of Wales P.O. Box 78, Cardiff CF1 1XL, UK

Received March 15, 1985. Revised May 21, 1985. Accepted May 21, 1985.

Sub-cellular fractions, isolated from cauliflower mosaic virus (CaMV)-infected turnip protoplasts, are capable of syn-thesising CaMV DNA in vitro on an endogenous template and of reverse transcribing oligo dT-primed cowpea mosaic virus RNA. The activity was not detected in mock-inoculated protoplasts. In vitro-labelled DNA hybridized to single-stranded M13 clones complementary to the putative origins of (–) and (+) strand CaMV DNA synthesis and to restriction endonuclease fragments encompassing more than 90% of the CaMV genome. The synthesis of (–) and (+) strand DNA appeared asymmetric. The template(s) for in vitro CaMV DNA synthesis are in a partially nuclease-resistant form. Fractions capable of in vitro CaMV DNA synthesis contained CaMV RNA both heterogeneous and as discrete species; they also contained a range of different sizes of CaMV DNA. Several lines of evidence indicate that this range of in vitro-labelled CaMV DNA, extending from 0.6kb to 8.0kb in length, represents elongating (–) strand DNA. These are discussed in relation to their role as possible replicative intermediates


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