Construction of a shuttle vector containing a single O6-methylguanine: a probe for mutagenesis in mammalian cells

Abstract

A shuttle vector, pKE15, was constructed for investigating the mechanisms by which single carcinogen-DNA adducts induce mutations in mammalian cells. pKE15 contains the SV40 origin of replication, the neomycin resistance gene, SV40 polyadenylation sequences and the pML2 origin of replication. Transfection of pKE15 into CHO cells established the G418-resistant phenotype; the frequency of G418-resistant clones was approximately 10(-4), a value that is similar to those obtained with other SV40-based vectors expressing the neomycin resistance gene. A tetranucleotide containing O6-methylguanine, a DNA adduct formed by carcinogenic alkylating agents, was incorporated into a 4-base gap positioned in the center of a PstI site. The tetranucleotide containing the adduct was physically mapped to a 14-base-pair region of the shuttle vector that included the ligation target, the PstI site. It was incorporated approximately equally into either of the complementary strands of the shuttle vector. The ligation efficiency of the tetranucleotide into the gapped genome was approximately 100% and was independent of the concentration of tetranucleotide used at concentrations ranging over one order of magnitude. The potential applications of the site-specifically modified genome for establishing the mutagenic fate of O6-methylguanine in repair-proficient and -deficient CHO cells are discussed.