Non-homologous recombination between adenovirus and AcNPV DNA fragments in cell-free extracts from insect Spodoptera frugiperda nuclei.

In previous work, we have developed a cell-free system from nuclear extracts of hamster cells to study the mechanism of integrative recombination between adenovirus type 12 (Ad12) DNA and hamster cell DNA (Jessberger et al., 1989; Tatzelt et al., 1992). We have also demonstrated that in insect cells the left terminal fragment of Ad2 DNA can insert by non-homologous recombination into the 32.6 to 34.5 map unit (EcoRI-O) fragment and into other segments of Autographa californica nuclear polyhedrosis virus (AcNPV) DNA (Xiong et al., 1991). We have now imitated this recombination event in vitro by incubating the E1 fragment of Ad2 DNA and the EcoRI-O fragment of AcNPV DNA, both in the plasmid-cloned circular forms, with partly purified nuclear extracts from Spodoptera frugiperda insect cells. Proteins from these extracts have been fractionated by gel filtration. After the reextraction of DNA from the incubation mixture, recombinants generated in this cell-free system have been identified directly with the polymerase chain reaction (PCR) by using Taq polymerase and appropriate primers which are unique to either of the two reaction partners. The recombinants identified are all different. The results of control experiments argue against the possibility that unspecific reaction products might have been generated during PCR. Nucleotide sequence determinations in some of the recombinants localize the sites of genetic exchange between the two partners and assess the non-homologous nature of the reaction. The recombinants are characterized by the presence of short patch homologies at or close to the sites of linkage between the reaction partners, as described earlier in the hamster cell system (Tatzelt et al., 1992). The occurrence of recombinants in the cell-free system can also be demonstrated by a biological test in which potential recombinants are isolated by transfection into recA- strains of Escherichia coli.