MAPPING OF REPLICATION INITIATION SITES IN HUMAN RIBOSOMAL DNA BY NASCENT-STRAND ABUNDANCE ANALYSIS

New techniques for mapping mammalian DNA replication origins are neede d. We have modified the existing nascent-strand size analysis techniqu e (L. Vassilev and E. M. Johnson, Nucleic Acids Res. 17:7693-7705, 198 9) to provide an independent means of studying replication initiation sites. We call the new method nascent-strand abundance analysis. We co nfirmed the validity of this method with replicating simian virus 40 D NA as a model. We then applied nascent-strand abundance and nascent-st rand size analyses to mapping of initiation sites in human (HeLa) ribo somal DNA (rDNA), a region previously examined exclusively by two-dime nsional gel electrophoresis methods (R. D. Little, T. H. K. Platt, and C. L. Schildkraut, Mel. Cell. Biol. 13:6600-6613, 1993). Our results partly confirm those obtained by two-dimensional gel electrophoresis t echniques. Both studies suggest that replication initiates at relative ly high frequency a few kilobase pairs upstream of the transcribed reg ion and that many additional low-frequency initiation sites are distri buted through most of the remainder of the ribosomal DNA repeat unit.