Analysis of chromosome replication by a BrdU antibody technique

Abstract

Chromosome replication was studied without synchronization in human lymphocyte and amniotic cell cultures visualizing very short 5-bromodeoxyuridine (BrdU) pulses by an immunologic technique (BAT). The findings agree in general with those facts known from earlier BrdU staining techniques. The very high sensitivity of BAT was shown to allow the detection of replication in a band where 1 in 200 nucleotides is replaced by BrdU. The main observations are: though the replication patterns after BAT appear strange the bands correspond to those described by the Paris Conference (1971). At the beginning of the S-phase a stepwise onset of replication in only a subset of R-bands is confirmed. There is a considerable difference in the sensitivity between early and late S (SE and SL) for the detection of BrdU pulses. This difference probably reflects a different spatial arrangement of chromatin in R-bands as compared with G-bands below the level of cytogenetic analysis. The use of short pulses did not reveal any additional subdivision of SE or SL. The correspondence between chromosomal bands and replicon clusters is discussed briefly with respect to the different time they need for replication.

The gene-richest bands of human chromosomes replicate at the onset of the S-phase

“…Moreover, we compared our H3 isochores in situ hybridization with the very early replicating R bands of Fig. 4a of Vogel et al (1989). In this case as well, the correspondence among many H3 bands and the early replicating R bands is clearly visible (Fig.…”

“…Also, the previous results by Kim et al (1975) showed that the majority of R bands replicating in the first 30 min and corresponding to replication classes I, II, and III coincide with H3 + and H3* bands. Moreover, when the highest sensitivity detection of the BrdU-substituted DNA obtained by immunological technique was applied, it was shown that not all the R bands, but only a subset of them, begin to replicate at the onset of the S phase (Vogel et al, 1989). When Fig.…”

“…The right member of each pair of chromosomes shows the H3 isochore hybridization, whereas the left member shows the very early replicated R bands identified in Fig. 4a of Vogel et al (1989). Biotinylated H3 isochores were detected with avidin-FITC, and chromosomes were stained with propidium iodide.…”

“…It has been shown that R bands replicate early in the S phase, whereas G bands replicate late (Dutrillaux et al, 1975(Dutrillaux et al, , 1976Biemont et al, 1978;Dutrillaux and ViegasPéquinot, 1981;Camargo and Cervenka, 1982;Vogel et al, 1989;Drouin et al, 1991;Fetni et al, 1996; see also Drouin et al, 1994, for a recent review). A number of these results have been obtained by incorporating 5-bromodeoxyuridine (BrdU) into replicating DNA, which generates a G-band or an R-band pattern depending upon the period in S phase in which BrdU was incorporated and by the staining method used, such as FPG (fluorochrome-photolysis-Giemsa), acridine orange, or immunological staining with a BrdU antibody (Dutrillaux et al, 1976;Vogel et al, 1989;Drouin et al, 1990;Fetni et al, 1996).…”

High-Resolution Analysis of DNA Replication in Released Chromatin Fibers Containing 5-Bromodeoxyuridine

“…Even the use of very short BrdU pulses did not reveal any additional order of replication below that of the structural banding pattern (15). Fluorescence in situ hybridization (FISH) on yeast artificial chromosome (YAC) DNA fibers has revealed structures resembling replication intermediates (bubbles and forks) in human DNA cloned in yeast (14).…”