Productive infection of mouse cells with polyoma virus yields mainly two types of particles: Complete virions and empty capsids. Empty polyoma capsids have been shown to be capable of interacting with DNA, in vitro, to form what has been referred to as polyoma-like particles (PLP). The particles are stable in high concentrations of salt and contain DNA protected by the capsid against the action of pancreatic DNase. The development of PLP into a gene transfer vehicle is the subject of the investigations described in the present dissertation. The approach has been to first, characterize the process of PLP formation and second, determine whether the genetic information contained in a specific DNA fragment and assembled into PLP in vitro can be transferred to cells and subsequently be expressed. In terms of PLP characteristics, the experimental results described in this dissertation show that the DNA extracted from PLP is heterogeneous in size. It has a mean molecular weight of 1.2 x 10⁶ with a standard deviation of ±0.5 x 10⁶. In addition, analysis of PLP DNA with restriction endonucleases revealed that a specific primary sequence or higher order structure is not required for PLP formation. Either linear, circular or supercoiled polyoma DNA, as well as, single-stranded DNA, rRNA and the synthetic homopolymers poly(dA).poly(dT) and poly(dG).poly(dC) can be used for PLP formation. Transfer of genetic information by PLP has been accomplished by using a restriction fragment containing the transforming sequences of polyoma DNA as a model gene. This fragment of polyoma DNA, which consists of 1,831 base pairs (approximately 1.2 x 10⁶ daltons) and extends clockwise from the BclI site to the EcoRI site on the conventional polyoma map, causes the induction of the transformed phenotype in rat cells grown in culture. Infection of rat F111 cells by PLP, containing this DNA fragment, results in DNA-mediated oncogenic transformation of the cells as indicated by the formation of dense foci. This gene transfer activity of PLP is shown to be 50 to 150 times more efficient than the widely used calcium phosphate coprecipitation method of introducing DNA into mammalian cells.

The involvement of topoisomerase in DNA replication in a prokaryotic and a eukaryotic system was examined. The mechanism of in vitro DNA replication by an isolated replicative enzyme complex was also investigated. In bacteriophage T4 there is evidence that the type II topoisomerase coded for by the phage is involved in the initiation of replicative growing points. We have looked at the topological structure of the replicating T4 nucleoid by sedimentation of the DNA in neutral sucrose gradients containing various amounts of ethidium bromide. It was determined that at no time after infection does the replicating T4 DNA contain any large amount of negative superhelicity. The absence of the phage topoisomerase did not affect the topology of the nucleoid. It was therefore concluded that the role of the T4 topoisomerase in initiating DNA synthesis in T4 was not exerted at the level of the general topology of the replicating T4 DNA. An isolation procedure for the T4 topoisomerase for pursuance of further studies was also described. New mammalian topoisomerases were shown to be stimulated by epidermal growth factor (EGF) in two cultured fibroblast cell lines. Topoisomerase activity was seen first in the cell cytoplasm and subsequently in the nucleus. The peak of topoisomerase activity in the nucleus corresponded to the peak of EGF-induced DNA synthesis in the cells. At least a part of the topoisomerase activity stimulated by EGF was shown to be due to a type II topoisomerase by the ATP-dependence of the activity. The topoisomerase activity in the cytoplasm was shown to exist in a non-soluble, sedimentable form. Further characterization of the topoisomerase containing complex isolated from the cytoplasm was carried out. The complex was seen to be non-membrane bound and complex. DNA polymerase and nucleoside diphosphate kinase activities were also demonstrated to be contained within the complex. It was shown that this cytoplasmic complex was capable of in vitro DNA replication. Many parameters of the in vitro DNA replication reaction were examined. The process was seen to mimic in vivo replication in several ways. The complex was shown to not only be able to elongate DNA but to initiate replication through the creation of a replication bubble.