DNA synthesis in HeLa cell nuclei isolated in a non-aqueous medium.
Nuclei were isolated from synchronized HeLa cells in the S-phase by a modification of the non-aqueous method described by Kirsch et al. (Science (1970) 168, 1592-1595). The method involved lyophilization of the cells, homogenization in non-aqueous glycerol and centrifugation in a gradient of 0-35% (w/w) 3-chloro-1,2-propanediol in glycerol. Such nucleic incorporated deoxyribonucleotides into DNA when incubated in an aqueous buffer containing Mg2+, ATP, dATP, dGTP, dCTP and dTTP. The product was sensitive to DNAase and banded with bulk DNA in isopycnic centrifugation. Sedimentation of the product in alkaline sucrose gradients after labelling of the nuclei for 2 min revealed labelled material in the 5 S peak and in the 18 S area. The material in the 5 S peak moved into the 12 S area after a 13 min chase.