title: Fractionated nuclear extracts from hamster cells catalyze cell-free recombination at selective sequences between adenovirus DNA and a hamster preinsertion site.
abstract: We have explored the mechanism of adenovirus type 12 (Ad12) DNA integration because of its importance for viral oncogenesis and as an example of insertional recombination. We have used a fractionated cell-free system from nuclear extracts of hamster cells and have partly purified nuclear proteins that could catalyze in vitro recombination. As recombination partners, the 20,880- to 24,049-nucleotide Pst I D fragment of Ad12 DNA and the hamster preinsertion sequence p7 from the Ad12-induced tumor CLAC1 have proven to recombine at higher frequencies than randomly selected adenoviral or cellular DNA sequences. A preinsertion sequence might carry elements essential in eliciting recombination. Patch homologies between the recombination partners seem to play a role in the selection of sites for recombination in vivo and in the cell-free system. Nuclear extracts from BHK21 cells were prepared by incubating the nuclei in 0.42 M (NH4)2SO4 and fractionated by Sephacryl S-300 gel filtration, followed by chromatography on Mono S and Mono Q columns. The purified products active in recombination contained a limited number of different protein bands, as determined by polyacrylamide gel electrophoresis and silver staining. The most highly purified fraction IV had helicase and topoisomerase I activities. We used two different methods to assess the in vitro generation of hamster DNA-Ad12 DNA recombinants upon incubation with the purified protein fractions: (i) transfection of the recombination products into recA- strains of Escherichia coli and (ii) the polymerase chain reaction by using amplification primers unique for each of the two recombination partners. In p7 hamster DNA, the nucleotide sequence 5'-CCTCTCCG-3' or similar sequences served repeatedly as a preferred recombination target for Ad12 DNA in the tumor CLAC1 and in five independent cell-free recombination experiments.
authors.affiliation: Institute of Genetics, University of Cologne, Germany.
authors.forename: J
authors.initials: J
authors.lastname: Tatzelt
authors.forename: K
authors.initials: K
authors.lastname: Fechteler
authors.forename: P
authors.initials: P
authors.lastname: Langenbach
authors.forename: W
authors.initials: W
authors.lastname: Doerfler
mesh_terms: D000256:Adenoviridae / Q000235:genetics*; D000818:Animals; D001483:Base Sequence; D002467:Cell Nucleus / Q000378:metabolism*; D002472:Cell Transformation, Viral; D002474:Cell-Free System; D006224:Cricetinae; D004279:DNA, Viral / Q000235:genetics; D066298:In Vitro Techniques; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides / Q000737:chemistry; D016133:Polymerase Chain Reaction; D011995:Recombination, Genetic
chemical_list: D004279:DNA, Viral; D009838:Oligodeoxyribonucleotides