Interphase chromosomes

Some fluorescent DNA stains can also be used for chromosome counterstaining, for detection of hybridized metaphase or interphase chromosomes in fluorescence in situ hybridization assays or for identifying apoptotic cells in cell populations (http //probes.invitrogen.com/handbook/sections/0806.html). For instance, Vybrant Apoptosis Assay Kit 4 (Molecular Probes) detects apoptosis on the basis of changes that occur in the permeability of cell membranes. This kit contains ready-to-use solutions of both YO-PRO-1 and propidium iodide nucleic acid stains. YO-PRO-1 stain selectively passes through the plasma membranes of apoptotic cells and labels them with moderate green fluorescence. Necrotic cells are stained red-fluorescent with propidium iodide. 

Marshall WF, Straight A, Marko JF, Swedlow J, Dernburg A, Belmont A, Murray AW, Agard DA, Sedat JW (1997) Interphase chromosomes undergo constrained diffusional motion in living cells. Cutr Biol 7 930-939... 

Transcription regulators are also known to be sumoylated. One of tbe early studies of tbis phenomenon showed that promyelocytic leukemia protein (PML) is a substrate for SUMO conjugation. Once SUMO is attached to the PML protein it is directed to a subdomain of tbe nucleus called tbe PML oncogenic domain (POD). It is thought that POD localization of the PML protein allows it to recruit other proteins such as transcription factors. Transcription factors in the POD can activate or inhibit transcription. Another transcription factor known to be sumoylated is Sp3. ° SUMO also has roles in chromatin condensation and interphase chromosome organization. ... 

R. K. Sachs, G. van den Engh, B. Trask, H. Yokota, and J. E. Hearst, A random-walk/giant-loop model for interphase chromosomes. Proc. Natl. Acad. Sci. USA 92, 2710-2714 (1995). 

J. Ostashevsky, A polymer model for the structural organization of chromatin loops and minibands in interphase chromosomes. Mol. Biol. Cell 9, 3031-3040 (1998). 

D. Zink, T. Cremer, R. Saffrich, R.Pischer, M. P.Trendelenburg, W. Ansorge, andE. H. Stelzer, Structure and dynamics of human interphase chromosome territories in vivo. Hum. Genet. 102, 241-251 (1998). 

Belmont, A.S., Braunfeld, M.B., Sedat, J.W., and Agard, D.A. (1989) Large-scale chromatin structural domains within mitotic and interphase chromosomes in vivo and in vitro. Chromosoma 98(2), 129-143. 

Trask, B.J., Allen, S., Massa, H., Fertitta, A., Sachs, R., van den Engh, G., and Wu, M. (1993) Studies of metaphase and interphase chromosomes using fluorescence in situ hybridization. Cold Spring Harb. Symp. Quant. Biol. 58, 767-775. 

The effects of chromosome structure on gene regulation in eukaryotes have no clear parallel in prokaryotes. In the eukaryotic cell cycle, interphase chromosomes appear, at first viewing, to be dispersed and amorphous (see Figs 12-41, 24-25). Nevertheless, several forms of chromatin can be found along these chromosomes. About 10% of the chromatin in a typical eukaryotic cell is in a more condensed form than the rest of the chromatin. This form, heterochromatin, is transcriptionally inactive. Heterochromatin is generally associated... 

For mapping, BrdU is added to enhance chromosome elongation and banding for metaphase chromosomes and to help distinguish G1 from S and G2 phase nuclei in the case of interphase chromosomes. Treatment of the cells for a few minutes with hypotonic solutions to make them swell and exposure to low temperatures, thus interfering with the stability of spindle fibres, can improve the preparations. 

A FIGURE 10-24 Model for the packing of chromatin and the chromosome scaffold in metaphase chromosomes. In interphase chromosomes, long stretches of 30-nm chromatin loop out from extended scaffolds. In metaphase chromosomes, the scaffold is folded further into a highly compacted structure, whose precise geometry has not been determined. 

A EXPERIMENTAL FIGURE 10-25 Fluorescent-labeled probes hybridized to interphase chromosomes demonstrate chromatin loops and permit their measurement. In situ hybridization of interphase cells was carried out with several different probes specific for sequences separated by known distances in linear, cloned DNA. Lettered circles represent probes. Measurement of the distances between different hybridized probes, which could be distinguished by their color, showed that some sequences (e.g.. A, B, and C), separated from one another by millions of base pairs, appear located near one another within nuclei. For some sets of sequences, the measured distances in nuclei between one probe (e.g., C) and sequences successively farther away initially appear to increase (e.g., D, E, and F) and then appear to decrease (e.g., G and H). The measured distances between probes are consistent with loops ranging in size from 1 million to 4 million base pairs. [Adapted from H. Yokota et al., 1995, J. Cell Biol. 130 1239.]... 

In certain cells of the fruit fly Drosophila melanogaster and related insects, interphase chromosomes are reduplicated 10 times, generating polytene chromosomes that are visible in the light microscope (see Figure 10-31). 

A network of intermediate filaments is often found as a laminating layer adjacent to a cellular membrane, where it provides mechanical support. The best example is the nuclear lamina along the inner surface of the nuclear membrane (see Figure 21-16). This supporting network is composed of lamin A and lamin C filaments cross-linked into an orthogonal lattice, which is attached by lamin B to the inner nuclear membrane through interactions with a lamin B receptor, an IFAP, in the membrane. Like the membrane skeleton of the plasma membrane, the lamin nuclear skeleton not only supports the inner nuclear membrane but also provides sites where nuclear pores and interphase chromosomes attach. Thus, the nuclear lamins organize the nuclear contents from the outside in. 

Fig. 1. Hierarchy of chromatin folding in the nucleus, during interphase, chromosomes are spread in a diffuse form and the nucleoli appear as dense structures. During metaphase chromosomes which consist of condensed sections of chromatin are formed. Chromatin in interphase nuclei and in the condensed sections of chromosomes contains looped domains which are formed by the 30 nm chromatin fibre. In vitro it is possible to further unfold the 30 nm fibre to a 3-D zig-zag structure revealing the nucleosomes that consist of histone HI, the core particle and the linker DNA...

Finally, it should be mentioned that a polymer model for the structural organization of chromatin loops in interphase chromosomes was developed by Oslashevsky (1998) on the basis of isochores. 

For several reasons, the interphase chromosome scaffold remains the most controversial of karyoskeletal elements. For example, it is currently uncertain whether the scaffold is one contiguous element, several discrete elements, or an in vitro artifact without biological significance. Nevertheless, roles for the scaffold in DNA replication, transcription, splicing, and (perhaps) mRNA export have been proposed. In the current context, it is critical to distinguish the interphase scaffold, referred to herein, from the mitotic or metaphase scaffold, studied extensively by others [for a review, see Saitoh and Laemmli (1993) and references therein]. 

Drosophila polypeptides have been definitively and specifically identified in subnuclear fractions enriched for all karyoskeletal elements they have been specifically localized to these elements in situ. Hence, the Drosophila nuclear lamina contains lamin Dmo derivatives (Smith et al., 1987 McConnell et al., 1987 Gruenbaum et al., 1988), lamin C (Bossie and Sanders, 1993 Riemer et al., 1995), and otefin (Hard et al., 1989). Nuclear pore complexes contain gp210o (McConnell etal, 1987 Berrios etai, 1995) as well as polypeptides with immunochemical homology to myosin (Berrios and Fisher, 1986 Berrios et al., 1991 Berrios, 1992). The interphase chromosome scaffold contains large amounts of the enzyme, DNA topoisomerase II (Berrios et al, 1985 McConnell et al, 1987 Meller et al, 1995). 

---