Figure 1.

Permeabilization of the nuclear envelope is required for replication of Xenopus erythrocyte nuclei by egg extract. (A) Nuclei were prepared by treating erythrocytes with LPC or SLO, and nuclear envelope integrity was determined by incubating nuclei with TRITC-labeled IgG (Tritc-IgG). Total DNA (DNA) was stained with Hoechst 33258. (B) Permeable (LPC) and intact (SLO) nuclei were incubated in egg extract, supplemented with [α-³²P]dATP, for various times as indicated. DNA replication is expressed as a percentage of incorporated label in the permeable sample at 10 h. The mass of DNA synthesized in this sample was 0.89 ng/μl of extract, representing ∼30% of the input DNA. (C) Permeable and intact nuclei were incubated in extract supplemented with 20 μM biotinylated dUTP for 4 h. Nuclei were isolated and stained for total DNA (DNA) and with Texas Red-streptavidin to detect biotin-dUTP incorporation into nascent DNA (Biotin). A representative field of nuclei is shown. (D) Two hundred nuclei from each sample in C were examined for Texas Red fluorescence. The percentages of biotin-labeled nuclei are shown.