This project was undertaken to study site specific mutagenesis in mammals. Mouse L cells (tk-/-) were co-transformed with double stranded RF DNA from PhiX174 am3 CS70 (am3 - amber mutation; CS70 - cold temperature sensitive mutation) and Lambda-phage containing the genomic tk+/+ gene. The transformed cells were selected on HAT medium and single colony isolates were made. One cell culture, T15-1.3, was analyzed for its content of X174 DNA by digestion with restriction enzymes, electrophoresis, southern blot, and probing with nick translated PhiX174. Several bands were visible, the strongest band co-migrated with PhiX174 DNA. The level of radioactivity indicated approximately 3-10 copies of PhiX174 doubled stranded DNA in this band. The DNA from the transformed L-cells were digested with the restriction enzyme pst-1 and ligased with T4 ligase. This DNA was transfected into E. coli spheroplasts and PhiX174 plaques were obtained from the transformed L-cells.