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

Nucleic Acids Res. 1985 Jun 25;13(12):4557-76. doi: 10.1093/nar/13.12.4557.

Abstract

Sub-cellular fractions, isolated from cauliflower mosaic virus (CaMV)-infected turnip protoplasts, are capable of synthesising 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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Cloning, Molecular
  • DNA / analysis
  • DNA Replication
  • DNA Restriction Enzymes
  • DNA, Viral / genetics*
  • Molecular Weight
  • Mosaic Viruses / genetics*
  • Nucleic Acid Hybridization
  • Plants / microbiology
  • Protoplasts / metabolism
  • RNA-Directed DNA Polymerase / metabolism*
  • Species Specificity
  • Templates, Genetic

Substances

  • DNA, Viral
  • DNA
  • RNA-Directed DNA Polymerase
  • DNA Restriction Enzymes