Discontinuous hairpin DNAs synthesised in vivo following specific and non-specific priming of cauliflower mosaic virus DNA (+) strands

Abstract

We have analysed, by two-dimensional gel electrophoresis, a population of cauliflower mosaic virus (CaMV) linear DNA forms from infected tissue which contain one or two discontinuities in the plus (+) strand at sites close to the putative priming sequences for (+) strand synthesis (designated G2(+), G3(+)). The proximal ends of the linear DNAs mapped to a region near the origin of DNA (−) strand synthesis at the tRNA primer binding site. The distal end of each linear DNA appeared to comprise heterogeneous single-strand extensions of the (−) strand extending from G2(+) and G3(+) in the direction of reverse transcription. However, strand-specific probes revealed that the ‘single-strand’ extensions contained sequences with both (+) and (−) strand polarity and their single-stranded sizes were twice that expected had they been double-stranded. This suggested that some extensions were hairpin structures which we confirmed by observing their rapid renaturation following transfer to nitrocellulose. We conclude that the linear discontinuous hairpin DNAs arose in vivo by interrupted reverse transcription of RNA templates followed by non-specific self-primed (+) strand synthesis. Generation of ‘full-length’ hairpins was prevented in some molecules by specific priming of (+) strands at G2(+) and G3(+), and in others, extended hairpin formation probably proceeded as a result of loss of specific (+) strand primer RNA fragments at G2(+) and G3(+). The significance of these DNA forms in an understanding of CaMV replication is discussed.