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 DNA from mammalian cells and a PCR-based assay has been used in order to compare the in vivo activities of DNA replication origins in different cell lines. Conventional PCR was firstly applied to detect the chromosomal activities of several known (origins associated with c-myc, hsp70, beta-globin, immunoglobulin mu-chain enhancer) and putative DNA replication origins (autonomously replicating sequences obtained from enriched libraries of human origins of DNA replication from normal and transformed cells) in four human cell lines (HeLa, NSF, WI-38 and SK-MG-1). Then, in nascent DNA samples from normal skin fibroblast (NSF) and HeLa cells, abundance of DNA sequences in the regions of five of these origins was determined by competitive PCR. Our results suggest that autonomously replicating sequences NOA3, S14, S3 and F15 are associated with functional chromosomal origins of replication. Quantitative comparison of origin activities demonstrates that origins associated with c-myc and NOA3 are approximately twice as active in HeLa cells as in NSF cells. The described approach can facilitate the identification of origins which may be differentially active in normal cells and transformed cells or in different cell types.
Tao
L
L
McGill Cancer Centre, McGill University, Montreal, Quebec, H3G 1Y6, Canada.
Nielsen
T
T
Friedlander
P
P
Zannis-Hadjopoulos
M
M
Price
G
G
D002461:Cell Line, Transformed; D004261:DNA Replication; D005347:Fibroblasts / Q000378:metabolism*; D005914:Globins / Q000235:genetics; D018840:HSP70 Heat-Shock Proteins / Q000235:genetics; D006367:HeLa Cells; D006801:Humans; D007148:Immunoglobulin mu-Chains / Q000235:genetics; D034882:Lamins; D009687:Nuclear Proteins / Q000235:genetics; D016133:Polymerase Chain Reaction; D016271:Proto-Oncogene Proteins c-myc / Q000235:genetics; D018741:Replication Origin; D012867:Skin / Q000166:cytology; D014407:Tumor Cells, Cultured
D018840:HSP70 Heat-Shock Proteins; D007148:Immunoglobulin mu-Chains; D034882:Lamins; D009687:Nuclear Proteins; D016271:Proto-Oncogene Proteins c-myc; D005914:Globins