To

Metaphase chromosome

1400 nm

Interphase extended scaffold-

associated chromatin

Interphase extended scaffold-

associated chromatin

Condensed scaffold-associated chromatin pgll

700 nm

Chromosome scaffold

300 nm

30-nm chromatin fiber of packed nucleosomes

"Beads-on-a-string" form of chromatin

Short region of DNA double helix

Chromosome scaffold

30-nm chromatin fiber of packed nucleosomes

"Beads-on-a-string" form of chromatin

300 nm

30 nm

11 nm

2 nm

30 nm

11 nm

2 nm

▲ 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.

In situ hybridization experiments with several different fluorescent-labeled probes to DNA in human interphase cells support the loop model shown in Figure 10-24. In these experiments, some probe sequences separated by millions of base pairs in linear DNA appeared reproducibly very close to one another in interphase nuclei from different cells (Figure 10-25). These closely spaced probe sites are postulated to lie close to specific sequences in the DNA, called scaffold-associated regions (SARs) or matrix-attachment regions (MARs), that are bound to the chromosome scaffold. SARs have been mapped by digesting histone-depleted chromosomes with restriction enzymes and then recovering the fragments that are bound to scaffold proteins.

In general, SARs are found between transcription units. In other words, genes are located primarily within chromatin loops, which are attached at their bases to a chromosome scaffold. Experiments with transgenic mice indicate that in some cases SARs are required for transcription of neighboring genes. In Drosophila, some SARs can insulate transcription units from each other, so that proteins regulating transcription of one gene do not influence the transcription of a neighboring gene separated by a SAR.

Loop of 30-nm chromatin fiber

Loop of 30-nm chromatin fiber

▲ 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.]

▲ EXPERIMENTAL FIGURE 10-26 During interphase human chromosomes remain in specific domains in the nucleus. Fixed interphase human lymphocytes were hybridized in situ to biotin-labeled probes specific for sequences along the full length of human chromosome 7 and visualized with fluorescently labeled avidin. In the diploid cell shown here, each of the two chromosome 7s is restricted to a territory or domain within the nucleus, rather than stretching throughout the entire nucleus. [From P Lichter et al., 1988, Hum. Genet. 80:224.]

Individual interphase chromosomes, which are less condensed than metaphase chromosomes, cannot be resolved by standard microscopy or electron microscopy. Nonetheless, the chromatin of interphase cells is associated with extended scaffolds and is further organized into specific domains. This can be demonstrated by the in situ hybridization of interphase nuclei with a large mixture of fluorescent-labeled probes specific for sequences along the length of a particular chromosome. As illustrated in Figure 10-26, the bound probes are visualized within restricted regions or domains of the nucleus rather than appearing throughout the nucleus. Use of probes specific for different chromosomes shows that there is little overlap between chromosomes in interphase nuclei. However, the precise positions of chromosomes are not reproducible between cells.

Chromatin Contains Small Amounts of Other Proteins in Addition to Histones and Scaffold Proteins

The total mass of the histones associated with DNA in chro-matin is about equal to that of the DNA. Interphase chro-

matin and metaphase chromosomes also contain small amounts of a complex set of other proteins. For instance, a growing list of DNA-binding transcription factors have been identified associated with interphase chromatin. The structure and function of these critical nonhistone proteins, which help regulate transcription, are examined in Chapter 11. Other low-abundance nonhistone proteins associated with chromatin regulate DNA replication during the eukaryotic cell cycle (Chapter 21).

A few other nonhistone DNA-binding proteins are present in much larger amounts than the transcription or replication factors. Some of these exhibit high mobility during electrophoretic separation and thus have been designated HMG (high-mobility group) proteins. When genes encoding the most abundant HMG proteins are deleted from yeast cells, normal transcription is disturbed in most other genes examined. Some HMG proteins have been found to bind to DNA cooperatively with transcription factors that bind to specific DNA sequences, stabilizing multiprotein complexes that regulate transcription of a neighboring gene.

Eukaryotic Chromosomes Contain One Linear DNA Molecule

In lower eukaryotes, the sizes of the largest DNA molecules that can be extracted indicate that each chromosome contains a single DNA molecule. For example, the DNA from each of the quite small chromosomes of ,S. cerevisiae (2.3 X 105 to 1.5 X 106 base pairs) can be separated and individually identified by pulsed-field gel electrophoresis. Physical analysis of the largest DNA molecules extracted from several genetically different Drosophila species and strains shows that they are from 6 X 107 to 1 X 108 base pairs long. These sizes match the DNA content of single stained metaphase chromosomes of these Drosophila species, as measured by the amount of DNA-specific stain absorbed. The longest DNA molecules in human chromosomes are too large (2.8 X 108 base pairs, or almost 10 cm long) to extract without breaking. Nonetheless, the observations with lower eukaryotes support the conclusion that each chromosome visualized during mitosis contains a single DNA molecule.

To summarize our discussion so far, we have seen that the eukaryotic chromosome is a linear structure composed of an immensely long, single DNA molecule that is wound around histone octamers about every 200 bp, forming strings of closely packed nucleosomes. Nucleosomes fold to form a 30-nm chromatin fiber, which is attached to a flexible protein scaffold at intervals of millions of base pairs, resulting in long loops of chromatin extending from the scaffold (see Figure 10-24). In addition to this general chromosomal structure, a complex set of thousands of low-abundance regulatory proteins are associated with specific sequences in chromosomal DNA.

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