A Fraction of Newly Synthesized DNA of HeLa Cells

HeLa cells were labelled for 10 or 30 minutes with [ 3 H]thymidine and the DNA fractionated with chloroform‐isoamylalcohol; almost 90% of the labelled DNA remained in the interphase whilst about 60% of total DNA (bulk) was extracted in the aqueous phase. When added to a column of methylated serum albumin adsorbed on kieselguhr the extracted DNA was eluted as a major labelled peak with 0.8 M NaCl but a small fraction could only be eluted at higher NaCl concentrations. This “tail” contained both bulk and recently labelled DNA. After a short pulse the specific activity of the DNA eluted at higher salt concentrations increased; this was confirmed when pooled fractions were concentrated on a caesium sulphate gradient. Electron microscopy and sucrose gradient centrifugation of fractions recovered on a Cs 2 SO 4 gradient shows that the eluted DNA was polydisperse (peak at about 18 S) with molecular dimensions of a few to almost 20 μ. When [ 3 H]uridine was used as a label, both labelled DNA and rapidly labelled RNA could be studied simultaneously in the same way: these experiments indicated that in the fractions analysed, the radioactivity due to RNA and to DNA was not carried on the same molecules, suggesting the mutual exclusion of replication and transcription.