biotin-16-dutp and bromodeoxyuridine-triphosphate

HeLa cells synchronized at different stages of the cell cycle were permeabilized and incubated with analogues of nucleotide triphosphates; then sites of incorporation were immunolabeled with the appropriate fluorescent probes. Confocal microscopy showed that sites of replication and transcription were not diffusely spread throughout nuclei, reflecting the distribution of euchromatin; rather, they were concentrated in 'foci' where many polymerases act together. Transcription foci aggregated as cells progressed towards the G1/S boundary; later they dispersed and became more diffuse. Replication was initiated only at transcription sites; later, when heterochromatin was replicated in enlarged foci, these remained sites of transcription. This illustrates the dynamic nature of nuclear architecture and suggests that transcription may be required for the initiation of DNA synthesis.

Topics: Autoantigens; Biotin; Cell Cycle; Cell Nucleus; Deoxyuracil Nucleotides; DNA; DNA Replication; HeLa Cells; Humans; Image Processing, Computer-Assisted; Ribonucleoproteins, Small Nuclear; RNA; snRNP Core Proteins; Transcription, Genetic