To study the organization of DNA replication in mammalian rRNA genes, the sites of initiation of DNA synthesis in rat and human rRNA genes were mapped by two independent techniques. In rat cells the growth of the nascent DNA chains was blocked by Trioxsalen cross-links introduced in vivo. The fraction of ``restricted'' nascent DNA chains labeled in vivo was isolated, and the abundance in this fraction of cloned ribosomal DNA sequences was determined by hybridization. In the experiments with human cells, the nascent DNA chains were allowed to grow unrestricted for a certain period of time and the movement of the replication forks along the rRNA genes was followed by hybridization of cloned ribosomal DNA sequences to the ``unrestricted'' nascent DNA fragments fractionated according to size. The results show that in both rRNA genes there are two well defined regions of initiation of DNA synthesis. The first one is located upstream of the transcription units and the second one is located at the 3`-end of the coding regions of the ribosomal DNA repeats.

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