Detection and analysis of UV-induced mutations in mammalian cell DNA using a λ phage shuttle vector

Abstract

In order to study mutagenesis in mammalian cells, stable mouse L-cell lines were established with multiple copies of a λ phage vector that contains the supF gene of Escherichia coli as a target for mutagenesis. Rescue of viable phage from high molecular weight mouse cell DNA using λ in vitro packaging extracts was efficient (5 phage per μg of cell DNA per copy) and yielded a negligible background of mutant phage (0 out of 54,605). From mouse cells exposed to 254-nm ultraviolet light (12 J/m2), 78,510 phage were rescued, of which 8 were found to have mutant supF genes. DNA sequence analysis of the mutants suggests that the primary site of UV mutagenesis in mammalian cells is at pyrimidine-cytosine (Y-C) sequences, and that the most frequent mutation at this site is a C→T transition.