PERMEABLE ANIMAL-CELL PREPARATION FOR STUDYING MACROMOLECULAR-SYNTHESIS - DNA-SYNTHESIS AND ROLE OF DEOXYRIBONUCLEOTIDES IN S-PHASE INITIATION

A method for selectively permeabilizing Chinese hamster ovary cells with lysolecithin is described. These cells retained their general morphology, intact organelles; 100% of their DNA and 75% of their total protein. They synthesized protein, RNA and DNA when supplied with appropriate substrates and cofactors, many of which do not penetrate intact cells. These permeable cells will be useful for studying the roles of nonpenetrating molecules such as substrates, effectors, and inhibitors of cellular metabolic processes, including macro-molecular syntheses, under relatively physiological conditions. As an initial study, some properties of DNA synthesis were investigated. DNA synthesis proceeded at an initial rate at least as high as in intact cells and continued for at least 2h, and 20% of the genome could be replicated. The DNA was first made as small (4S) pieces that were rapidly ligated to 4-21 X 106 dalton DNA. Normal, semiconservative synthesis was demonstrated by showing increased density following BrdUTP incorporation. Washing the permeable cells shortened the duration of DNA synthesis; adding back the solution in which the permeable cells were prepared restored the initial ability. A soluble factor thus seems needed to allow continued DNA synthesis. The cells do not stop making DNA because of temperature-dependent deterioration or because of exhaustion of added substrates. The ability of a synchronized culture approaching S to make DNA appeared at the same time, and not earlier, in permeable cells supplied with the deoxyribonucleoside triphosphates, as in intact cells. Hence supplying these precursors cannot alone be the final event needed for initiation of the S period. A supply of deoxyribonucleotides is necessary for DNA synthesis; hydroxyurea blocked DNA synthesis by intact cells, but the capacity of these cells to synthesize DNA was restored by permeabilizing the cells and supplying deoxyribonucleotides. Ribonucleotide reductase thus does not appear to have a sufficient role for initiation of the S period, but only a necessary one. © 1978, American Chemical Society. All rights reserved.