The basic structural unit of chromatin, the nucleosome, was described by Roger Kornberg in 1974. Two types of experiments led to Kornberg's proposal of the nucleosome model. First, partial digestion of chromatin with micrococcal nuclease (an enzyme that degrades DNA) was found to yield DNA fragments approximately 200 base pairs long. In contrast, a similar digestion of naked DNA (not associated with proteins) yielded a continuous smear of randomly sized fragments. These results suggested that the binding of proteins to DNA in chromatin protects regions of the DNA from nuclease digestion, so that the enzyme can attack DNA only at sites separated by approximately 200 base pairs. Consistent with this notion, electron microscopy revealed that chromatin fibers have a beaded appearance, with the beads spaced at intervals of approximately 200 base pairs. Thus, both the nuclease digestion and the electron microscopic studies suggested that chromatin is composed of repeating 200-base-pair units, which were called nucleosomes.
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More extensive digestion of chromatin with micrococcal nuclease was found to yield particles (called nucleosome core particles) that correspond to the beads visible by electron microscopy. Detailed analysis of these particles has shown that they contain 146 base pairs of DNA wrapped 1.65 times around a histone core consisting of two molecules each of H2A, H2B, H3, and H4 (the core histones) (Figure 4.9). One molecule of the fifth histone, H1, is bound to the DNA as it enters each nucleosome core particle. This forms a chromatin subunit known as a chromatosome, which consists of 166 base pairs of DNA wrapped around the histone core and held in place by H1 (a linker histone).
The packaging of DNA with histones yields a chromatin fiber approximately 10 nm in diameter that is composed of chromatosomes separated by linker DNA segments averaging about 80 base pairs in length (Figure 4.10). In the electron microscope, this 10-nm fiber has the beaded appearance that suggested the nucleosome model. Packaging of DNA into such a 10-nm chromatin fiber shortens its length approximately sixfold. The chromatin can then be further condensed by coiling into 30-nm fibers, the structure of which still remains to be determined. Interactions between histone H1 molecules appear to play an important role in this stage of chromatin condensation.
Electron micrograph of an interphase nucleus. The euchromatin is distributed throughout the nucleus. The hetero-chromatin is indicated by arrowheads, and the nucleolus by an arrow. (Courtesy of Ada L. Olins and Donald E. Olins, Oak Ridge National Laboratory.)
The extent of chromatin condensation varies during the life cycle of the cell. In interphase (nondividing) cells, most of the chromatin (called euchromatin) is relatively decondensed and distributed throughout the nucleus (Figure 4.11). During this period of the cell cycle, genes are transcribed and the DNA is replicated in preparation for cell division. Most of the euchromatin in interphase nuclei appears to be in the form of 30-nm fibers, organized into large loops containing approximately 50 to 100 kb of DNA. About 10% of the euchromatin, containing the genes that are actively transcribed, is in a more decondensed state (the 10-nm conformation) that allows transcription. Chromatin structure is thus intimately linked to the control of gene expression in eukaryotes, as will be discussed in Chapter 6.
In contrast to euchromatin, about 10% of interphase chromatin (called heterochromatin) is in a very highly condensed state that resembles the chromatin of cells undergoing mitosis. Heterochromatin is transcriptionally inactive and contains highly repeated DNA sequences, such as those present at centromeres and telomeres.
Scanning electron micrograph of metaphase chromosomes. Artificial color has been added. (Biophoto Associates/Photo Researchers Inc.)
An electron micrograph of DNA loops attached to the protein scaffold of metaphase chromosomes that have been depleted of histones. (From J. R. Paulson and U. K. Laemmli, 1977. Cell 12: 817.)
As cells enter mitosis, their chromosomes become highly condensed so that they can be distributed to daughter cells. The loops of 30-nm chromatin fibers are thought to fold upon themselves further to form the compact metaphase chromosomes of mitotic cells, in which the DNA has been condensed nearly 10,000-fold (Figure 4.12). Such condensed chromatin can no longer be used as a template for RNA synthesis, so transcription ceases during mitosis. Electron micrographs indicate that the DNA in metaphase chromosomes is organized into large loops attached to a protein scaffold (Figure 4.13), but we currently understand neither the detailed structure of this highly condensed chromatin nor the mechanism of chromatin condensation.
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(A) A light micrograph of human chromosomes spread from a metaphase cell. (B) Human chromosomes are arranged in pairs numbered from the largest (chromosome 1) to the smallest. The chromosomes shown are from a female, so there are 22 pairs of autosomes and two X chromosomes. (Craig Holmes/Biological Photo Service.)
Metaphase chromosomes are so highly condensed that their morphology can be studied using the light microscope (Figure 4.14). Several staining techniques yield characteristic patterns of alternating light and dark chromosome bands, which result from the preferential binding of stains or fluorescent dyes to AT-rich versus GC-rich DNA sequences. These bands are specific for each chromosome and appear to represent distinct chromosome regions. Genes can be localized to specific chromosome bands by in situ hybridization, indicating that the packaging of DNA into metaphase chromosomes is a highly ordered and reproducible process.