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.
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.
C M
CM
Thomas
R
R
Hull
J A
JA
Bryant
A J
AJ
Maule
D003001:Cloning, Molecular; D004247:DNA / Q000032:analysis; D004261:DNA Replication; D004262:DNA Restriction Enzymes; D004279:DNA, Viral / Q000235:genetics*; D008970:Molecular Weight; D009029:Mosaic Viruses / Q000235:genetics*; D009693:Nucleic Acid Hybridization; D010944:Plants / Q000382:microbiology; D011523:Protoplasts / Q000378:metabolism; D012194:RNA-Directed DNA Polymerase / Q000378:metabolism*; D013045:Species Specificity; D013698:Templates, Genetic
D004279:DNA, Viral; D004247:DNA; D012194:RNA-Directed DNA Polymerase; D004262:DNA Restriction Enzymes