DIFFERENTIAL DNA-REPLICATION ORIGIN ACTIVITIES IN HUMAN NORMAL SKIN FIBROBLAST AND HELA-CELL LINES

A modification of the extrusion method for the isolation of nascent DN A from mammalian cells and a PCR-based assay has been used in order to compare the in vivo activities of DNA replication origins in differen t cell lines. Conventional PCR was firstly applied to detect the chrom osomal activities of several known (origins associated with c-myc, hsp 70, beta-globin, immunoglobulin mu-chain enhancer) and putative DNA re plication origins (autonomously replicating sequences obtained from en riched libraries of human origins of DNA replication from normal and t ransformed cells) in four human cell lines (HeLa, NSF, WI-38 and SK-MG -1). Then, in nascent DNA samples from normal skin fibroblast (NSF) an d HeLa cells, abundance of DNA sequences in the regions of five of the se origins was determined by competitive PCR. Our results suggest that autonomously replicating sequences NOA3, S14, S3 and F15 are associat ed with functional chromosomal origins of replication. Quantitative co mparison of origin activities demonstrates that origins associated wit h c-myc and NOA3 are approximately twice as active in HeLa cells as in NSF cells. The described approach can facilitate the identification o f origins which may be differentially active in normal cells and trans formed cells or in different cell types. (C) 1997 Academic Press Limit ed.