Nucleic Acids Research, 1991, Vol. 19, No. 9 2325-2330
© 1991
MOLECULAR BIOLOGY |
DNA forms of the geminivirus African cassava mosaic virus consistent with a rolling circle mechanism of replication
Department of virus Research, John Innes Institute, John Innes Centre for Plant Science Research Colney Lane, Norwich NR4 7UH, UK
*To whom correspondence should be addressed
Received February 8, 1991. Accepted April 2, 1991.
We have analysed DNA from African cassava mosaic virus (ACMV) infected Nicotlana benthamlana by twodimensional agarose gel electrophoresls and detected ACMV-speclflc DNAs by blot-hybridisation. ACMV DNA forms Including the previously characterised singlestranded, open-circular, linear and supercoiled DNAs along with five previously uncharacterised heterogeneous DNAs (H1 - H5) were resolved. The heterogeneous DNAs were characterised by their chromatographlc properties on BND-cellulose and their ability to hybridise to strand-specific and doublestranded probes. The data suggest a rolling circle mechanism of DNA replication, based on the sizes and strand specificity of the heterogeneous single-stranded DNA forms and their electrophoretic properties in relation to genome length single-stranded DNAs. Second-strand synthesis on a single-stranded virussense template is evident from the position of heterogeneous subgenomic complementary-sense DNA (H3) associated with genome-length virus-sense template (VT) DNA. The position of heterogeneous virus-sense DNA (H5), ranging in size from one to two genome lengths, Is consistent with its association with genome-length complementary-sense template (CT) DNA, reflecting virus-sense strand displacement during replication from a double-stranded intermediate. The absence of subgenomic complementary-sense DNA associated with the displaced virus-sense strand suggests that replication proceeds via an obligate single-stranded intermediate. The other species of heterogeneous DNAs comprised concatemeric singlestranded virus-sense DNA (H4), and double-stranded or partially single-stranded DNA (H1 and H2).
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