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© The Rockefeller University Press, 0021-9525/1998//975 $5.00
The Journal of Cell Biology, Volume 140, Number 5, , 1998 975-989

Article

Interphase Cell Cycle Dynamics of a Late-Replicating, Heterochromatic Homogeneously Staining Region: Precise Choreography of Condensation/Decondensation and Nuclear Positioning



Gang Li, Gail Sudlow, and Andrew S. Belmont

Department of Cell and Structural Biology, University of Illinois, Urbana-Champaign, Urbana, Illinois 61801

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 ~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, ~0.3-µm-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.

Abbreviations used in this paper: BrdU, bromodeoxyuridine; DAPI, 4,6-diamidino-2-phenylindole; GFP, green fluorescent protein; HSR, homogeneously staining region; NE, nuclear envelope; NLS, nuclear localization signal.

Address all correspondence to Dr. Andrew S. Belmont, Department of Cell and Structural Biology, B107 Chemical and Life Sciences Building, 601 South Goodwin Avenue, University of Illinois, Urbana-Champaign, Urbana, IL 61801. Tel.: (217) 244-2311. Fax: (217) 244-1648. E-mail: asbel{at}uiuc.edu


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