Non-random incorporation of 5-bromodeoxyuridine in rat cell DNA

Abstract

Secondary cultures of rat embryo cells were exposed for 24 hrs. to 10- 7M [ 3H] thymidine (TdR) or 10 −7M [ 3H]5-bromodeoxyuridine (BrdU) in order to localize and compare the distribution of the isotopes in DNA. DNA was extracted, sheared, and centrifuged to equilibrium through neutral and alkaline CsCl density gradients. The DNA band from each gradient type was separated into a “heavy” and “light” fraction, and DNA-DNA reassociation hybridizations were performed on each sample. Renaturation profiles revealed that each fractionated DNA sample was representative of the complete rat cell genome, except for the “light” [ 3H]BrdU-DNA prepared by centrifugation through alkaline CsCl gradients. This fraction was predominantly depleted of labeled late repetitive and intermediate sequences. Uncentrifuged rat DNA was sequentially fractionated during reassociation into rapidly, intermediate, and slowly reassociating sequences by hydroxyapatite chromatography. Relative specific activities of each component revealed a non-uniform distribution of [ 3H]BrdU moieties as compared to [ 3H]TdR. These results suggest a nonrandom incorporation of 10 −7M BrdU into rat cell DNA sequences.