Fig. 3.

Tipin is not essential for replication. (A) Mock- or Tipin-depleted extracts were supplemented with sperm nuclei (3,000 nuclei per microliter) and cy3-dATP (Amersham). After 30 min, 3 μl was spotted on a microscope slide and mixed with a fixative containing Hoechst 33258 (blue fluorescence). Red fluorescence due to cy3-dATP incorporation indicates a replicating nucleus. (B) Sperm nuclei (1,000 nuclei per microliter) were added to an interphase Xenopus extract (mock- or Tipin-depleted). Replication was labeled with [α-³²P]dATP from 0 to 30 min, from 30 to 60 min, from 60 to 90 min, or from 90 to 120 min from nuclei addition. Replicated DNA was purified, loaded on agarose gel, and analyzed by autoradiography.