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.

We have shown that a commercially available antibody allows the demonstration of bromodeoxyuridine (BrdU) incorporation into mammalian chromosomes by the typical immunologic staining techniques. The replication patterns obtained are comparable to those after BrdU-incorporation and FPG-staining, though with reversed staining specificity. The quality of the patterns equals that of commonly used differential staining but the sensitivity of BrdU-detection is superior. Thus the new technique may be very promising for all cytogenetic studies concerning any kind of chromosome replication.

A modified immunologic technique is described for the purpose of demonstrating replication patterns on mammalian chromosomes after partial histone depletion. Replication patterns were induced by BrdU substitution and visualized by BrdU antibodies, coupled with peroxidase (diaminobenzidine/H₂O₂ or immunogold-detection. The replication patterns obtained by this technique did not reveal any additional details of replication compared to those shown by conventional cytogenetic staining. However, the possibility of demonstrating replication patterns on these partially histone-depleted chromosomes may prove useful for chromosomal in situ hybridization studies since the chromosomes produced are considerably larger than those seen in conventional preparations.