Integration Sites for Moloney Murine Leukemia Virus DNA in Infected Mouse Cells

Abstract

The integration of viral DNA in mouse cells infected with Moloney murine leukemia virus (M-MuLV) has been studied by the “blotting” technology introduced by Southern (1975). We were interested in determining if there is a single site in the chromosomal DNA at which M-MuLV viral DNA is integrated, a small number of sites, or a large number of possible integration sites. The technique used involved cleavage of infected cell DNA with a sequence-specific restriction endonuclease, resolution of the resulting DNA fragments by electrophoresis in 0.6% agarose gels, and blot transfer of the separated fragments to nitrocellulose filters. Detection of DNA fragments with sequence homology to M-MuLV was achieved by hybridization with 32P-labeled M-MuLV complementary DNA (cDNA). The labeled cDNA (approximately 2 x 108 cpm/µg) was synthesized in an endogenous reaction using purified virus and added calf thymus oligo-deoxynucleotide primers in order to generate a uniformly representative probe (Fan and Verma, 1978).