BrdU pulse/reverse staining protocols for investigating chromosome replication.
By using a reverse Giemsa staining procedure (TT chromatin pale, TB chromatin dark) it is possible to detect replication in metaphase chromosomes with short (approximately 10 min) 5-bromodeoxyuridine (BrdU) pulses. A pulse protocol allows us to consider the question "What is replicating at this point in time?" and we have investigated replication patterns during cycle transit in stimulated human female lymphocytes. A clear-cut demarcation between R-zone early and G-zone late was not found. Instead, whilst replication commences (with a very staggered start) in R-zones, activity soon appears to transgress band boundaries and gives rise to cells with unclassifiable patterns where chromosomes take on a mottled or reticulate appearance. Replication in R-zones dies out leaving a clear G-zone pattern persisting for the remainder of S which terminates with a very staggered finish. When pulse duration is increased (approximately 1 h) the frequency of unclassifiable cells falls and occasional "mixed-pattern" cells appear which have, within the same cell, typical R- and G-zone regions. The existence of such cells indicates that if a mid-S replication pause exists (and the absence of any mid-S wave of pale stained cells suggests that it does not) it does not make exclusive separation between dark R- and G-band zones.
Aghamohammadi
S Z
SZ
Division of Cell and Molecular Biology, Medical Research Council Radiobiology Unit, Chilton, Didcot, UK.
Savage
J R
JR
D001973:Bromodeoxyuridine / Q000378:metabolism*; D002478:Cells, Cultured; D002875:Chromosomes / Q000378:metabolism*; D004261:DNA Replication; D005260:Female; D006801:Humans; D007621:Karyotyping; D008214:Lymphocytes; D013194:Staining and Labeling
D001973:Bromodeoxyuridine