Single-Strand DNA Shuttle Vectors for Analyzing DNA-Damage Processing

Abstract

Mutagenesis is a major biological process giving rise to both positive action (such as genetic variability) and deleterious effects (such as aging and carcinogenesis). To understand the mechanism of mutagenesis, it is necessary to accumulate data on DNA sequence changes produced by spontaneous or induced DNA lesions. Much information on various base sequence changes has been obtained in bacteria, but it has been more difficult to produce similar spectra in mammalian cells. One way to resolve the technical difficulty imposed by the extreme complexity of the cellular genome is to use bacterial target genes on shuttle vectors able to replicate in both mammalian and bacterial cells (1). Indeed, shuttle vectors are hybrid plasmids containing DNA sequences derived from the bacterial plasmid pBR322 and from a mammalian virus, so that they can replicate both in bacteria and in eukaryotic cells. Three functional DNA sequences are present (Fig. 1):a viral replication origin and eventually specific information required for replication in mammalian cells, a bacterial replication origin, and a drug resistance gene to permit replication and maintenance in bacteria. These features allow a given shuttle vector to be processed, i.e., replicated and repaired, in the mammalian cell environment. The resulting plasmids are easily recovered and maintained in E. coli. Finally, bacterial target genes present in these vectors facilitate the screening of the occasional alterations (Fig. 1).