Isolation of short interspersed repetitive DNA sequences present in the regions of initiation of mammalian DNA replication

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Abstract
Nascent DNA chains containing the putative replication origins were isolated from cells of human embryonic lung fibroblasts, HeLa, Ehrlich ascites tumor and Guerin ascites tumor as described earlier. The synthesis of these nascent chains correlated with the ability of cells to initiate semiconservative DNA replication. Reassociation and electrophoretic analysis showed that the nascent chains from all 4 cell lines contained middle repetitive DNA in the form of short interspersed sequences. Mouse repetitive sequences were isolated and hybridized to Escherichia coli, chicken, calf and rat DNA and to homologous hn[heterogeneous nuclear]RNA. The kinetics of hybridization indicated that the repetitive sequences found in the vicinity of the replication origins were order-specific and were not heavily transcribed. Reassociation experiments, in which homologous DNA isolated from nuclei digested with micrococcal nuclease to different extents was used as a driver, showed that these repetitive sequences were organized into nucleosomes like the bulk of the chromatin.