Replication banding

Replication banding

A G- or R-banding pattern can be produced on chromosome preparations if they are grown in the presence of the thymidine analogue 5-bromo-2-deoxyuridine (BrdU) and stained with the fluorescent dye Hoechst 33258.

Hoechst 33258 also show that its fluorescence is enhanced by AT-rich DNA while the enhancement is less with GC-rich DNA.
This method utilizes the fact that different regions of the chromosome complement replicate at different stages during S phase.

Replication banding

By controlling the timing of when the pulse of BrdU is administered to cell cultures relative to the harvest time, either an early- or late-replicating banding pattern can be produced. If BrdU is added at culture initiation, but removed about 6 hours before harvest at 48 hours, a G-banding pattern is obtained.
Early GC rich Euchromatin Lightly stain / Fluoresce Dully Late AT rich Heterochromatin Darkly stain / Fluoresce Brightly

Replication banding

If BrdU is only added about 6 hours before cultures are harvested, R-banding results.
Early GC rich Euchromatin Darkly stain / Fluoresce brightly Late AT rich Heterochromatin Lightly stain / Fluoresce Dully

Fig. Metaphase spread after replication banding. This preparation was obtained from blood cultures to which BrdU was added 6 hours before harvesting. Chromosomes show an Rbanding pattern. In this instance, the normal X is late-replicating. With late-pulse BrdU cultures utilized, the late-replicating X stains lightly (arrowed)

Early Replication banding (T-Pulse)

When BrdU is present at culture initiation, it is present for the early part of one cycle of DNA replication, and therefore chromosome regions which replicate early in S phase (R-bands/Euchromatin) become BrdUsubstituted and are therefore weakly fluorescing or light-staining with Giemsa. The G-bands/Heterochromatin replicate late in S phase, after the BrdU has been removed from the cultures, and therefore incorporate thymidine and are brightly fluorescing, or dark-staining with Giemsa (Gbanding).

Late Replication banding (B-pulse)

Alternatively, if BrdU is only added to cultures about 6 hours before harvesting, it is only present for the late part of one DNA replication cycle and the earlyreplicating regions (R-bands) have already been replicated, incorporating thymidine, and fluoresce brightly or stain darkly with Giemsa. Only the late-replicating regions (G-bands) become BrdU-substituted and therefore fluoresce weakly or stain lightly with Giemsa (R-banding).

Replication banding

Homologous chromosomes show similar replication patterns But, in females the early- and late replicating X chromosomes can be distinguished. The late-replicating X chromosome (Heterochromatin) is darkly stained with early pulse BrdU replication banding and lightly stained with late-pulse BrdU replication banding.

Fluorochrome-photolysisGiemsa (FPG) staining

FLUORESCENCE PLUS GIEMSA (FPG) STAINING

Replication banding

If cells undergo two complete cycles of replication in the presence of BrdU, the sister chromatids of each chromosome are differentially stained. Enabling the detection of sister chromatid exchanges (SCEs).

Replication banding

BrdU is a thymidine analogue that is readily incorporated into chromosomes. When chromosomes are stained with the fluorochrome Hoechst 33258 it binds to AT-rich region, chromosome regions that replicate in the presence of BrdU contain BrdU-substituted DNA hence doesnot gets stained.

FPG staining

Replication banding

SCE preparations are stained with the fluorochrome Hoechst 33258. They are then either viewed by fluorescence microscopy (360400 nm) or, if they are first exposed to UV light and then stained with Giemsa stain [ Fluorochrome-photolysisGiemsa (FPG) staining], they can be analysed using light microscopy.

FPG staining technique (fluorochrome photolysis- Giemsa)

Hoechst 33258 and light causes photlysis of BrdU in DNA and a series of radical reactions take place resulting in the reduce affinity of chromosome regions containing BrdU rich DNA for the Giemsa stain. Consequently, only the DNA regions which do not incorporate BrdU will be stained in chromosome.

FPG staining

Applications

Alternative methods for G- and R- banding. Replication banding is useful for investigating the pattern of X-inactivation in females carrying structural abnormalities involving the X chromosome (for nonrandom event). The production of harliquinized chromosome staining patterns enables the detection of SCEs. Normal human cells show a mean frequency of 58 SCEs/cell, but SCE levels have been demonstrated to increase with exposure to many mutagens.

Applications

SCE has proved more sensitive for the detection of chromosome damage than measurement of chromosomal aberrations. This method is also used as a diagnostic test for the chromosome instability disorder Bloom syndrome, in which cells show a significant increase from the normal baseline level of spontaneous SCEs. SCEs can also be used to study cell kinetics by indicating the duration of the various stages of the cell cycle.