Essential nucleotides in the polyomavirus origin region

Abstract

We have isolated a series of point mutants in the polyomavirus origin-intergenic control region by using a procedure which exploits the single-stranded nature of DNAs cloned into M13 phage both for the generation of mutants and for their analysis by DNA sequencing. In this report we describe the effects of cytosine X guanine----thymine X adenine base-pair substitutions in the polyomavirus origin region upon replication in mouse 3T6 cells of the M13-polyomavirus constructs. Our results indicate that sequences near the center of a 34-base-pair sequence with dyad symmetry are important for replication, whereas specific nucleotides near the ends of the dyad symmetry are not important. Furthermore, a putative large T antigen-binding site at nucleotides 60 to 80 can be mutated without affecting replication function as measured in this assay.