Local mutagenesis: a method for generating viral mutants with base substitutions in preselected regions of the viral genome

Abstract

DNA from simian virus 40 (SV40) was prepared for local mutagenesis by nicking the molecule at a specific site with a restriction endonuclease that recognizes one site in SV40 DNA and then extending the nick enzymatically to expose a short, single-stranded segment of DNA. The "gapped" DNA was treated with a single-strand-specific mutagen, sodium bisulfite, which converts cytosine to uracil. After mutagenesis, the gap was repaired with DNA polymerase, generating molecules resistant to the restriction enzyme used to make the initial nick. From cells infected with DNA thus modified, SV40 mutants were isolated that had enzyme-resistant genomes. In some cases, precise positions of G.C to A.T transitions could be inferred from the patterns of susceptibility of mutant DNA to other restriction endonucleases whose recognition sequences were altered by the mutagenesis procedure. One of the restriction endonuclease sites mutagenized (Bgl I) maps at the origin of SV40 DNA replication and near sequences corresponding to the 5' ends of viral mRNAs. Many of the resulting Bgl I-resistant mutants yielded small plaques, suggesting partial defectiveness in DNA replication or transcription.