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Intact nuclei are defined by the exclusion of large fluorescently labeled molecules that lack a nuclear localization signal (Fig 1) Damaged nuclei are per-meable to such molecules
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CHOC 400 is a CHO cell line that contains ∼1000 tandemly integrated copies of a 243-kb segment of DNA harboring the DHFR gene, the 2BE2121 gene, and a 55-kb intergenic region that were amplified by stepwise selection in methotrexate (4). The pattern of initiation of replication within the amplified locus is indistinguishable from that within the wild-type DHFR locus (70).
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84920294958
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6 nuclei per milliliter were stained with DAPI (1 μg/ml) and affinity-purified, Texas red-labeled IgG (150 μg/ml) (Jackson Laboratory). Photographs were taken with a Nikon Labophot-2 fitted with a 100× Plan lens (numerical aperture, 1.25) and were composed with Photoshop software.
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32P]deoxyadenosine 5′-triphosphate (25 μCl/ml). 6-DMAP was added to 3 mM. The percentage of labeled nuclei was determined by indirect immunofluorescence after incubation in extracts containing 250 μM bromodeoxyundine triphosphate. Similar results were obtained in four separate experiments, although the length of the lag period (10 to 30 min) and the total amount of DNA replicated varied with the batch of extract.
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We thank B. Knox, D. Dimitrova, M. DePamphilis, J. Huberman, and B. Burhans for critical reading of the manuscript and G. Wahl and G. Leno for helpful advice. Supported by grants to D M G. from the Hendricks Foundation, the Milheim Foundation, and the National Science Foundation (MCB-9505907).
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