Semiconservative DNA replication in vitro I. Properties of two systems derived from mouse P-815 cells by permeabilization or lysis with Brij-58

Poly(ADP‐ribose) polymerase activity in nucleotide permeable cells

“…In this experiment all three samples were exposed to 500 rad y-radiation immediately prior to loading onto the gradient [7]. This confirms the observation that the DNA is not fragmented in the permeabilized cells [3] .…”

“…Incubation of the permeabilized cells at 26'C for 90 min in the presence of the complete DNA synthesis mixture [3] prevented both the DNA fragmentation ( fig.SC) and the increase in poly(ADP-ribose) polymerase activity ( fig.2). We do not yet know whether we are preventing degradation of DNA in these conditions, or whether we have established an in vitro DNA repair system.…”

“…The modification [3] of the procedure [4] was used. Cells were cooled to O"C, washed with Pucks saline A (5.4 mM KC1 (400 mg/litre), 137 mM NaCl (8 g/litre), 4.2 mM NaHCOs (350 mg/litre) and 5.5 mM glucose (1 g/litre)) and resuspended in hypotonic solution at 2-3 X 10' cells/ml for permeabilization.…”

“…DNA synthesis in permeabilized cells is a semiconservative continuation of the in vivo synthesis [3]. Using this system the activity of poly-(ADP-ribose) polymerase is very low.…”

Maturation of Parvovirus LuIII in a Subcellular System. I. Optimal Conditions for in vitro Synthesis and Encapsidation of Viral DNA

“…At 15 h post-infection (or at the time indicated) the infected, prelabelled monolayer cultures were washed twice with an isotonic phosphate buffer containing 0-1 ~ trypsin, 0.137 M-NaC1, 2.7 mM-KCl and 0.54 mM-EDTA and then incubated for 1 min at 37 °C. Cells were detached and suspended in a modified MEM , washed once with Puck saline A (140 mM-NaCl, 5.4 mM-KC1, 0.1~ glucose, 0.03 ~ N aHCO3) and resuspended in ice-cold lysis solution at a density of about 107 cells/ml (Reinhard et al, 1977). The lysis solution consisted of 40 mM-HEPES pH 7-8, 80 mM-KCI, 2 mM-dithiothreitol (DTT), 2~ (w/v) dextran, 300 mi-sucrose, l mM-EGTA, 4 mM-MgCl2, 2 mM-ATP and contained 0.01 ~ Brij-58.…”