ON THE INSERTION OF FOREIGN DNA INTO MAMMALIAN GENOMES - MECHANISM AND CONSEQUENCES

We have studied the integration of adenovirus type 12 (Ad12) DNA in tr ansformed and hamster tumor cells over many years. Upon infection of h amster cells with Ad12, viral DNA has been found in association with h amster chromosomes, possibly in part integrated into the host genome. Ad12 DNA integration is not sequence specific. Transcriptionally activ e sites of the host genome show a preponderance for foreign DNA insert ion. We are pursuing the mechanism of Ad12 DNA integrative recombinati on in a cell-free system prepared from hamster cell nuclear extracts. In a number of Ad12-transformed hamster cell lines or in cell lines ca rrying foreign DNA, we have located the inserted Ad12 DNA copies on ha mster chromosomes by fluorescent in situ hybridization (FISH). Among t he consequences of Ad12 DNA integration, we have studied the de novo m ethylation of the integrated foreign (Ad12) DNA and increases in DNA m ethylation in several cellular genes and DNA segments in Ad12-transfor med and hamster tumor cells. Several lines of evidence argue for the n otion that parameters in addition to nucleotide sequence, in particula r site of integration and/or the chromatin configuration of the integr ated DNA, are important in generating de novo methylation patterns. Th e de novo methylation of integrated foreign DNA can be interpreted as an old cellular defense mechanism against the activity of foreign gene s in an established genome. Pursuing this concept, we have asked for t he most likely portal of entry of foreign DNA, supposedly the gastroin stetinal tract in most animals. This hypothesis has been tested by fee ding mice linearized or circular, double-stranded bacteriophage M13mp1 8 DNA. A small amount of this DNA transiently survives the digestive r egime of the animals' GI tract, although in a heavily fragmented form. A minute proportion of the fed M13mp18 DNA can be retrieved from the bloodstream of mice between 2 and 8 h after feeding, mainly associated with the leukocyte population.