Immunofluorescent visualization of DNA replication sites within nuclei of Chinese hamster ovary cells.
The spatial distribution of replication sites was studied by a sensitive method in cells cultured in vitro. Exponentially growing Chinese hamster ovary cells were permeabilized and pulse labeled in the presence of deoxyribonucleoside triphosphates, dTTP being replaced by biotin-11-dUTP as a substrate for DNA replication. The distribution of replication sites was visualized in isolated nuclei by fluorescent microscopy of samples taken periodically after short-term (2 min) in vitro labeling and pulse-chase experiments. Propidium iodide and 4,6-diamino-2-phenylindole served as fluorescent probes for total cellular DNA. Avidin-fluorescein isothiocyanate and biotinylated goat antiavidin antibody were used in an amplification procedure to fluorescently label the incorporated biotin-11-dUTP. Similar experiments using synchronized cells showed the distribution of replicons at different stages of S phase.
Banfalvi
G
G
Institute of Biochemistry Department I, Semmelweis University Medical School, Budapest, Hungary.
Wiegant
J
J
Sarkar
N
N
van Duijn
P
P
D000818:Animals; D002460:Cell Line; D002467:Cell Nucleus / Q000648:ultrastructure*; D002478:Cells, Cultured; D006224:Cricetinae; D003412:Cricetulus; D004261:DNA Replication; D005260:Female; D005434:Flow Cytometry; D008856:Microscopy, Fluorescence; D010053:Ovary; D012093:Replicon