DNA forms of the geminivirus African cassava mosaic virus consistent with a rolling circle mechanism of replication

Se analizo ADN de Nicotiana benthamiana infectada con CAMV mediante electroforesis bidimensional en geles de agarosa y se detectaron ADN especificos de CAMV mediante hibridacion en papel de filtro. Se resolvieron las formas de ADN de CAMV incluyendo los ADN monocatenarios, de circulo abierto, lineales y superenrollados caracterizados previamente, y 5 ADN heterogeneos (H1-H5) no caracterizados previamente. Los ADN heterogeneos se caracterizaron por sus propiedades cromatograficas en BND-celulosa y por su capacidad para hibridar en sondas bicatenarias y de cadena especifica. Los datos sugieren un mecanismo circular de duplicacion del ADN, que se basa en el tamano y en la especificidad de la cadena de las formas heterogeneas de ADN monocatenario y en sus propiedades electroforeticas en relacion con los ADN monocatenarios de longitud del genoma. La sintesis bicatenaria en un modelo de virus monocatenario se manifiesta en la posicion del ADN subgenomico heterogeneo complementario (H3) relacionado con el ADN del modelo del virus de longitud del genoma (VT). La posicion del ADN del virus heterogeneo (H5), cuyo tamano varia de una a dos longitudes del genoma, es compatible con su relacion con el ADN del modelo complementario de longitud del genoma (CT), lo cual refleja el desplazamiento de la cadena del virus durante la repeticion de un intermedio bicatenario. La ausencia de ADN complementario subgenomico relacionada con la cadena desplazada del virus sugiere que la repeticion ocurre a traves de un intermedio monocatenario obligado. Las demas especies de ADN heterogeneo comprendieron ADN monocatenario de virus concatemerico (H4) y ADN parcialmente monocatenario o bicatenario (H1 y H2). (RA-CIAT)

We have analysed DNA from African cassava mosaic virus (ACMV)- infected Nicotiana benthamiana by two-dimensional agarose gel electrophoresis and detected ACMV-specific DNAs by blot- hybridisation. ACMV DNA forms including the previously characterised single-stranded, open-circular, linear and supercoiled DNAs along with five previously uncharacterised heterogeneous DNAs (H1-H5) were resolved. The heterogeneous DNAs were characterised by their chromatographic properties on BND- cellulose and their ability to hybridise to strand-specific and double-stranded 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 virus- sense 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 single-stranded virus-sense DNA (H4), and double-stranded or partially single-stranded DNA (H1 and H2). (AS)