Isolation and characterization of virus-specific double-stranded DNA from tissues infected by bean golden mosaic virus

Abstract

A double-stranded (ds) DNA which may be a replication intermediate was isolated from bean (Phaseolus vulgaris L. "Top Crop") leaves systemically infected with bean golden mosaic virus, a whitefly-transmitted plant virus with a genome of circular single-stranded (ss) DNA. The isolation method used phenol/chloroform extraction, hydroxyapatite column chromatography, and rate-zonal centrifugation. The dsDNA had sequences complementary to those of viral DNA. The guanine-plus-cytosine content was 35%, and the sedimentation coefficient in alkaline sucrose density gradients was similar to that of viral ssDNA. Digestion of the dsDNA by Hha I endonuclease produced fragments that corresponded exactly in number and size with those produced by complete digestion of circular viral ssDNA by Hha I, when the fragments were denatured and analyzed on polyacrylamide gels. The dsDNA molecule was a circular structure with one discontinuity in one strand; hybridization results suggest that some of a the dsDNA has a discontinuity in the viral strand and some has a discontinuity in the nonviral strand. On the basis of these structures for the dsDNA, a preliminary model for replication of viral DNA is discussed.