Isolation and Partial Characterization of Different Defective DNA Molecules Derived from Polyoma Virus

Abstract

Supercoiled DNA molecules purified from mouse cells infected with high-multiplicity-passaged polyoma virus has a broader size distribution and sediments more slowly than DNA derived from low-multiplicity-passaged virus. The shorter DNA molecules are predominately noninfectious. Virus populations containing distinct size classes of defective virus DNA were isolated by growing virus from single cells infected by a defective and nondefective helper virus (infectious center). This technique probably results in the cloning of defective virus particles. By applying the infectious center method to DNA from various fractions of sucrose gradients it has been possible to obtain shorter circular DNA molecules ranging in size from 50 to 95% of the unit-length polyoma DNA molecule. The shorter molecules in any one preparation are homogeneous in size. This class size is retained upon repeated passage of crude viral lysates at high multiplicity. Thus far, all the purified shorter DNA molecules tested appear to be noninfectious and largely resistant to cleavage by the R 1 restriction enzyme. Some of the purified defective molecules have been found to interfere with the production of infectious virus upon co-infection with unit-length infectious polyoma DNA.