The spiroplasma virus 4 replicative form cloned in Escherichia coli transfects spiroplasmas

The replicative form (RF) of spiroplasma virus 4 (SpV4) has been purified from infected cells of Spiroplasma melliferum strain G1 by alkaline lysis followed by low melting point agarose gel electrophoresis. A partial restriction map has been established. The circular RF was linearized by cutting at the unique ClaI restriction site and has been cloned in Escherichia coli HB101 using the plasmid pBR328 as a vector. The recombinant plasmid was purified by equilibrium centrifugation in ethidium bromide-cesium chloride gradient. After ClaI endonuclease digestion, the inserted SpV4 RF DNA was recovered by low melting point agarose gel electrophoresis and was recircularized by ligation. The cloned SpV4 RF DNA was demonstrated to be infectious by transfection.