Construction in vitro of mutants of simian virus 40: insertion of a poly(dA·ddT) segment at the hemophilus parainfluenza II restriction endonuclease cleavage site

A biochemical procedure has been developed for the construction of covalently closed circular double-stranded DNA containing a short segment of poly(dA·dT) inserted at a specifie site. In particular, we have prepared circular simian virus 40 DNA with a short (about 50 base-pairs) insertion of poly(dA·dT) at the site cleaved by the Hemophilus parainfluenza II restriction endonuclease (0.735 map position). The insertion interrupts the normal nucleotide sequence and alters the viral phenotype. Circular SV40(I) DNA was cleaved with the HpaII endonuclease to form unit-length, double-stranded linear molecules; the exposed 5′-ends were trimmed back by λ 5′-exonuclease digestion; short extensions of poly(dA) or poly(dT) were added to the exposed 3′ hydroxyl ends with terminal deoxynucleotidyl transferase; an equimolar mixture of the poly(dA) and poly(dT)-ended linear DNAs was denatured and then annealed to reform duplex structures; and the resulting hydrogen-bonded circular molecules were covalently sealed in the presence of DNA polymerase I and DNA ligase. The viral DNA containing the insertion at 0.735 map position is infectious without a helper virus, although plaques produced on monolayers of monkey kidney cells (CV-IP) appeared later and were much smaller than those produced by wild-type DNA. At least a portion of the inserted poly(dA·dT) sequence is maintained through repeated cycles of viral growth in monkey cells, and can be located in the viral DNA by a modification of standard electron microscopic heteroduplex techniques. The method should be applicable for the preparation of insertion mutants in any infectious circular double-stranded DNA, depending on the availability of endonucleases capable of cleaving the DNA at single unique sites.