A new type of insertion mutation in monkey cells: insertion accompanied by long target site duplication.

We have developed a system for the detection of a new type of insertion mutation in mammalian cells. We have used a shuttle vector, plasmid pNK1, which contains the SV40 and pBR322 replication origins, and ApR, galK, and neoR genes. This plasmid was introduced into monkey COS1 cells, allowed to replicate, and then recovered plasmids were reintroduced into Escherichia coli HB101 to detect insertion mutations in the galK gene. We selected galK− KMR ApR mutants in order to eliminate galK− KmS deletion mutants. Insertion mutations in the plasmids recovered were then screened by agarose gel electrophoresis. Finally, insertion mutants that had the following characteristics were selected. First, they had the ability to produce gal+ revertants caused by the precise excision of inserted DNA in E. coli, implying that they had a target site duplication on both sides of the insertion. Second, they contained some repetitive sequence(s) as judged by hybridization with a bulk monkey DNA probe. Nucleotide sequence analysis of one of the mutants, 15K-1, showed that it contained α-satellite sequences within the coding region of the galK gene. It contained \(13\frac{1}{2}\) tandem repeat units of α-satellite sequence and was flanked by a 64 bp target site duplication, indicating that the α-satellite sequence had been translocated from the monkey genome into the plasmid by illegitimate recombination. Another insertion mutant, N11-1, contained an 11 kb insert which included an unknown repetitive sequence that was also flanked by a target site duplication of 353 bp. Since both of the insertion mutations contain long target site duplications, we concluded that the insertion mutations detected here are a new type of insertion mutation. A model for the formation of the insertion-duplication mutation is proposed, in which DNA replication plays a role in this illegitimate recombination.