Deoxyribonucleic acid synthesis in isolated nuclei from baby-hamster kidney cells (BHK-21/C13). Characterization of the system

Abstract

A comparative study of some commonly employed laboratory procedures for studying DNA synthesis in isolated nuclei was carried out. Nuclei isolated from baby-hamster kidney (BHK-21/C13) cells synthesize DNA for 30-60min at 37 degrees C in a reaction requiring uni- and bi-valent cations, ATP and all four deoxyribonucleoside 5'-triphosphates. The addition of either ribonucleotides or cytosol from S-phase cells had no effect, but DNA synthesis was stimulated by some dextrans (mol.wt. 5x10(6)). The extent of synthesis was influenced by apparently minor variations in experimental conditions. For example, DNA synthesis by nuclei in Tris/HCl, pH7.5, was only 50% of that observed in Hepes/NaOH, pH7.5; the presence of detergents Triton X-100, Triton N-101, Nonidet P-40, Brij 58 and Tween 80 in the incubation medium altered the amount of synthesis to different extents. Although most detergents inhibited synthesis, a stimulation occurred with Tween 80 (150% of controls). These effects were reversed on washing the nuclei, except that of Brij 58, which inhibited DNA synthesis by 90-95% irreversibly. Anomalous sucrose-density-gradient sedimentation behaviour of the DNA, and of precursor [(3)H]-dTTP, was observed when nuclei were lysed with solutions of sodium dodecyl sulphate/Mg(2+) or with Sarkosyl/Mg(2+), but consistent results, showing that the DNA synthesized in vitro sedimented exclusively at about 4S, were obtained when nuclei were lysed with sodium dodecyl sulphate (without Mg(2+))/EDTA, digested with proteinase K and heated at 100 degrees C with 11% (v/v) formaldehyde to prevent macromolecular association. These results, coupled with density-labelling studies with bromodeoxyuridine and CsCl-density-gradient analysis, showed that DNA synthesis in these nuclei was replicative and was restricted to a covalent extension of Okazaki pieces previously initiated in vivo. No new initiations were observed, and the DNA was not ligated into larger molecules. The cessation of DNA synthesis after about 60 min was due to the complete utilization of available primer/template DNA.

Effect of drugs on deoxyribonucleic acid synthesis in isolated mammalian cell nuclei. Comparison with partially purified deoxyribonucleic acid polymerases

“…Our studies with nuclei isolated from BHK-21/C13 cells show that synthesis in vitro is limited to a covalent elongation of Okazaki pieces previously initiated in vivo' (Burke & Pearson, 1979). These DNA fragments were shown to be about 100-200 nucleotides long by sucrose-density-gradient and gel-electrophoretic techniques, and we did not detect any ligation of these into higher-molecular-weight DNA. Since this DNA synthesis, although limited, is well defined it provides an opportunity to determine the identity of the enzyme that catalyses this elongation process.…”

NAD+, ADP-ribosylation and transcription in permeabilized mammalian cells

“…Baby-hamster kidney cells (BHK-2 1/C 13; Macpherson & Stoker, 1962) were used. Cells were grown routinely at 37°C in an atmosphere of 02/C02 (19:1) in minimal Eagle's medium (Glasgow modification) containing 10% (w/v) tryptose phosphate broth and 10% (v/v) foetal-calf serum as previously described (Burke & Pearson, 1979).…”