Complementary strand-specific sequences from unique fragments of adenovirus type 2 DNA for hybridization-mapping experiments

Separation of the complementary strands of adenovirus type 2 DNA by poly(U,G)-CsCl density gradient centrifugation permitted studies of Ad2³ DNA renaturation with independently variable concentrations of each complementary strand. Single-stranded DNA was isolated by hydroxylapatite chromatography following exhaustive incubation under such conditions, and was found to selectively represent sequences of the complement present in excess during the incubation. This result was exploited in a general method for isolation of complementary strand-specific sequences of radioactively labeled Ad2 DNA or restriction enzyme fragments of Ad2 DNA. Liquid phase saturation-hybridization experiments were carried out with labeled DNA representing each complementary strand of the six endo R.EcoRI cleavage fragments of Ad2 DNA and unlabeled messenger RNA prepared from HeLa cells late after productive infections with Ad2. The results were combined with the known endo R.EcoRI cleavage map of Ad2 DNA to construct a low-resolution map showing physically separated regions, on both complementary strands of Ad2 DNA, represented in mRNA late after infection.