Interphase cell cycle dynamics of a late-replicating, heterochromatic homogeneously staining region: Precise choreography of condensation/decondensation and nuclear positioning

Recently we described a new method for in situ localization of specific DNA sequences, based on lac operator/repressor recognition (Robinett, C,C., A, Straight, G, Li, C, Willhelm, G, Sudlow, A, Murray, and A.S. Belmont. 1996, J. Cell Biol. 135:1685-1700). We have applied this methodology to visualize the cell cycle dynamics of an similar to 90 Mbp, late-replicating, heterochromatic homogeneously staining region (HSR) in CHO cells, combining immunostaining with direct in vivo observations, Between anaphase and early G1, the HSR extends approximately twofold to a linear, similar to 0.3-mu m-diam chromatid, and then recondenses to a compact mass adjacent to the nuclear envelope. No further changes in HSR conformation or position are seen through mid-S phase. However, HSR DNA replication is preceded by a decondensation and movement of the HSR into the nuclear interior 4-6 h into S phase, During DNA replication the HSR resolves into linear chromatids and then recondenses into a compact mass; this is followed by a third extension of the HSR during G2/prophase. Surprisingly, compaction of the HSR is extremely high at all stages of interphase, Preliminary ultrastructural analysis of the HSR suggests at least three levels of large-scale chromatin organization above the 30-nm fiber.