Differential transcription of early and late-replicating DNA in human cells

Abstract

Several studies suggest that most of the functional genes of mammalian cells are contained in the early-replicating sequences of DNA. There is little direct evidence, however, to support this view. To determine whether the extent of transcription of different DNA sequences is related to the order in which these sequences are replicated, I have selectively labelled early- and late-replicating DNA with [ 3H]thymidine ([ 3H]TdR) and determined the extent to which the 3H-labelled non-repeated sequences of each DNA preparation are hybridized by increasing concentrations of RNA from exponentially growing KB cells. The fraction of DNA hybridized at infinite RNA concentration was then estimated by a plotting method which linearizes the data. Selective labelling of DNA was achieved by synchronizing a culture of KB cells by a double block of DNA synthesis and then labelling portions of the culture 0–3 h (early) or 6–9 h (late) after release of the cells from the second block. At all RNA concentrations tested, the fraction of early-replicating DNA hybridized was significantly greater than that of late-replicating DNA. At infinite RNA concentration the value for early-replicating DNA was 3–4 times as great as that of late-replicating DNA. If it is assumed that the fraction of DNA hybridized at infinite RNA concentration is proportional to the fraction of DNA which is transcribed, it can be concluded that 3–4 times as much early-replicating DNA is transcribed as late-replicating DNA in exponentially growing KB cells.