Immunofluorescent visualization of DNA replication sites within nuclei of Chinese hamster ovary cells |
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| Authors: | G. Banfalvi J. Wiegant N. Sarkar P. van Duijn |
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| Affiliation: | (1) Institute of Biochemistry Department I, Semmelweis University Medical School, P.O. Box 260, H-1444 Budapest 8, Hungary;(2) Department of Cytochemistry and Cytometry, Leiden University, Wassenaarseweg 72, 2333 AL Leiden, The Netherlands;(3) Department of Metabolic Regulation, Boston Biomedical Research Institute, 02114 Boston, MA, USA |
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| Abstract: | Summary 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-pheny-lindole 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. |
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