Identification of chromosomal bands replicating early in the S phase of normal human fibroblasts

Normal human fibroblasts (NHF1) were released from confluence arrest (G(0)) and replated in medium containing bromodeoxyuridine (BrdU) and aphidicolin . Despite severe reduction in the rate of DNA synthesis by aphidicolin, cel ls reentering the cell cycle incorporated BrdU at regions of the human geno me that replicated very early in S phase, After removal of aphidicolin and BrdU from the tissue culture medium, cells were collected in mitosis. Q-ban ding with 4',6-diamidino-2-phenylindole/actinomycin D was used to identify metaphase chromosomes. A monoclonal anti-BrdU antibody and a fluorescein is othiocyanate (FITC)-conjugated goat anti-mouse antibody were used to identi fy the BrdU-labeled sites. The criterion for scoring DNA replication sites was the detection of FITC fluorescence at homologous regions of both sister chromatids. Early replicating regions mapped within R-bands, but not all R -bands incorporated BrdU. Chromosomal bands 1p36.1, 8q24.1, 12q13, 15q15, 1 5q22, and 22q13 were labeled in 53% or more of the copies of these chromoso mes in the data set, suggesting that these sites replicated very early in S phase. Chromosomal band 15q22 was the most frequently labeled site (64%), which indicates that it contains some of the earliest replicating sequences in normal human fibroblasts. (C) 1998 Academic Press.