Viable polyoma virus with four BamHI decanucleotide linkers inserted at the 26-minute HincII site.

BamHI decanucleotide linkers (5' CCGGATCCGG 3') were ligated to full-length linear polyoma DNA partially cleaved with HincII, and the recombinant DNA was transfected into mouse 3T6 cells. A viable virus (PYNB5) was isolated which contains four linkers at the 26-min HincII site. PYNB5 is encapsidated with a larger major viral capsid protein (VP1) as predicted from the DNA sequence. PYNB5 appears to have the same growth and physical properties as the wild-type virus with the exception of greater inactivation upon dilution of virus stocks with water. When PYNB5 DNA is cleaved with BamHI, the larger of the two resulting fragments (PY66) contains an intact early region of the virus, including the origin of DNA replication. PY66 complements a polyoma early region mutant for growth and may be useful as a cloning vector for foreign DNA.