Use of alkaline sucrose gradients in a zonal rotor to detect integrated and unintegrated avian sarcoma virus-specific DNA in cells

Abstract

We have attempted to distinguish integrated and unintegrated forms of avian sarcoma virus-specific DNA in cells by sedimentaton through an alkaline sucrose gradient in a slowly reorienting zonal rotor. Results obtained with this procedure are similar to those obtained by the more convenient analysis of networks of high-molecular-weight cell DNA. Most, if not all, viral DNA appears completely integrated into the host cell genome in an avian sarcoma virus-transformed mammalian cell and in normal chicken cells (in which viral DNA is genetically transmitted). Fully transformed duck cells and duck embryo fibroblasts infected for 20 to 72 h contain both integrated and unintegrated viral DNA; up to one copy per cell is integrated within 20 h after infection, and four to eight copies are integrated in fully transformed cells. The amount of unintegrated DNA varies but may comprise over 75% of the viral DNA in acutely infected cells and from 20 to 70% of the viral DNA in fully transformed cells. The unintegrated DNA in either case consists principally of duplexes with "minus" strands the length of a subunit of the viral genome (2.5 X 10(6) to 3 X 10(6) daltons) and relatively short "plus" strands (0.5 X 10(6) to 1.0 X 10(6) daltons).