5 Supplement

Abstract

Two recent developments—the molecular cloning of eukaryotic DNA in bacteria and site-specific mutagenesis in vitro—have provided a rapid way to construct and map mutants in the genomes of SV40 and polyoma virus. Since they are propagated in bacteria, it is now possible to obtain viral mutants that cannot be propagated in eukaryotic cells because they are cis-acting, trans-dominant, or are not complementable for other reasons.

So far two sets of such mutants have been obtained. The first was generated in a recombinant plasmid consisting of a unit-length SV40 DNA molecule cloned via its BamHI cleavage site into the Escherichia coli plasmid pMK16 (Gluzman et al. 1980a,b), The resulting recombinant contains only one site of cleavage for the restriction enzyme BglI, whose recognition site lies very close to the origin of SV40 DNA replication. By selecting in various ways for plasmids resistant to the action of BglI, a large number of mutants have been obtained that have suffered small deletions around the SV40 origin. They can be divided into two categories: those that produce T antigen and those that do not. The smallest deletions (4 nucleotides in length) destroy the function of the origin of replication. These findings are in good agreement with earlier results that placed the outer limits of the origin of DNA replication in a 75-nucleotide tract spanning the BglI restriction site (Gutai and Nathans 1978; Subramanian and Shenk 1978). It is known that point mutations within the BglI recognition site drastically affect the efficiency of...