BRDU PULSE REVERSE STAINING PROTOCOLS FOR INVESTIGATING CHROMOSOME-REPLICATION

被引:8
作者
AGHAMOHAMMADI, SZ
SAVAGE, JRK
机构
[1] Division of Cell and Molecular Biology, Medical Research Council Radiobiology Unit, Chilton, Didcot
关键词
D O I
10.1007/BF01737292
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
By using a reverse Giemsa staining procedure (TT chromatin pale, TB chromatin dark) it is possible to detect replication in metaphase chromosomes with short (~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 (~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. © 1990 Springer-Verlag.
引用
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页码:76 / 82
页数:7
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