Distribution of 5-bromodeoxyuridine and thymidine in the DNA of developing chick cartilage

Abstract

In order to study the mechanism of the irreversible effects of BrdUrd on the differentiation of limb bud mesenchyme to cartilage, the reannealing behavior of DNA obtained from such cells was examined. Cells incubated with [3H]thymidine ([3H]dThd) during days 1 and 2 of culture incorporated label into repetitive, moderately repetitive, and unique classes of DNA. In contrast, when 5-bromo-2'-[3H]deoxyuridine ([3H]Brd Urd) was added during the first 48 hr (in the presence of 32 muM BrdUrd), the label was preferentially incorporated into a late moderately repetitive region. Simultaneous incubation of unlabeled BrdUrd and [3H]dThd revealed a selective inhibition of [3H]dThd incorporation into moderately repetitive regions. Cultures incubated during days 3 and 4 with [3H]dThd incorporated label into all three classes of DNA; however, when [3H]dThd was present during days 3 and 4 in cultures previously incubated with BrdUrd during days 1 and 2, the [3H]dThd was incorporated preferentially in the late moderately repetitive region. The melting behavior of this reannealed DNA was identical with that of the reannealed 1-2 day [3H]BrdUrd-labeled, late moderately repetitive DNA. Turnover experiments revealed that whereas there was no loss of [3H]deoxycytidine or [3H]dThd, 37% of [3H]BrdUrd activity was lost from the DNA in 2 days after removal of the isotopes.