FINE MAPPING OF A REPLICATION ORIGIN OF HUMAN DNA

A highly sensitive procedure was developed for the identification of t he origin of bidirectional DNA synthesis in single-copy replicons of m ammalian cells. The method, which does not require cell synchronizatio n or permeabilization, entails the absolute quantification, by a compe titive PCR procedure in newly synthesized DNA samples, of the abundanc e of neighboring DNA fragments distributed along a given genomic regio n. This procedure was utilized for mapping the start site of DNA repli cation in a 13.7-kb region of human chromosome 19 coding for lamin B2, which is replicated immediately after the onset of S phase in HL-60 c ells. Within this region, DNA replication initiates in a 474-bp area c orresponding to the 3' noncoding end of the lamin B2 gene and the nont ranscribed spacer between this gene and the 5' end of another highly t ranscribed one, This localization was obtained both in aphidicolin-syn chronized and in exponentially growing HL-60 cells.