MAPPING THE SITES OF INITIATION OF DNA-REPLICATION IN RAT AND HUMAN RIBOSOMAL-RNA GENES

To study the organization of DNA replication in mammalian rRNA genes, the sites of initiation of DNA synthesis in rat and human rRNA genes w ere mapped by two independent techniques. In rat cells the growth of t he 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 ribos omal DNA sequences was determined by hybridization. In the experiments with human cells, the nascent DNA chains were allowed to grow unrestr icted for a certain period of time and the movement of the replication forks along the rRNA genes was followed by hybridization of cloned ri bosomal DNA sequences to the ''unrestricted'' nascent DNA fragments fr actionated according to size, The results show that in both rRNA genes there are two well defined regions of initiation of DNA synthesis. Th e first one is located upstream of the transcription units and the sec ond one is located at the 3'-end of the coding regions of the ribosoma l DNA repeats.