Polyoma DNA: a physical map

Abstract

The action of restriction enzymes on polyoma DNA was studied with uniformly (32)P-labeled viral DNA obtained from either infected 3T6 or secondary mouse-embryo cells. Three restriction enzymes were used to construct a physical map of the polyoma genome. An enzyme from Hemophilus parainfluenzae, Hpa(II), cleaved polyma DNA into eight unique fragments (Hpa(II)-1 to Hpa(II)-8), ranging in size from 27.3 to 1.8% of the genome. An enzyme from Hemophilus influenzae, Hin(III), gave two fragments (56 and 44%); and a third enzyme from Escherichia coli, EcoR(I), cut at a single unique site. The physical map of the polyma genome was constructed from methods involving: (1) further digestion of the fragments produced by enzymes EcoR(I) and Hin(III) with Hpa(II), and (2) analysis of the products of partial digestion with Hpa(II). Analysis by electron microscopy of replicating DNA molecules (less than 50% replicated) cut with the Hin(III) enzyme, in combination with other studies, has indicated that the origin of DNA replication is located at 71 +/- 3 map units from the EcoR(I) cleavage site, probably in Hpa(II)-5.