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Note: The sizes of H1, H2A, and H2B histones vary somewhat from species to species. The values given are for bovine histones. Source: Data are from A.L. Lehninger, D. L. Nelson, and M. M. Cox, Principles of Biochemistry, 3d ed. (New York: Worth Publishers, 1993), p. 924.

l'| At the simplest level is a double-stranded helical structure of DNA

DNA double helix l'| At the simplest level is a double-stranded helical structure of DNA

DNA double helix

300 nm

.that forms loops averaging 300 nm in length.

300 nm

QThe 300-nm fibers are I compressed and folded to J\k

' produce a 250-nm-wide fiber.

700 nm rt Tight coiling of the 250-nm fiber produces the chromatid of a chromosome.

700 nm

QThe 300-nm fibers are I compressed and folded to J\k

' produce a 250-nm-wide fiber.

rt Tight coiling of the 250-nm fiber produces the chromatid of a chromosome.

1400 nm

1400 nm

11.5 Chromatin has a highly complex structure with several levels of organization.

or kinks, in its helical structure as it winds around the histones.

The fifth type of histone, H1, is not a part of the core particle but plays an important role in the nucleosome structure. The precise location of H1 with respect to the core particle is still uncertain. The traditional view is that H1 sits outside the octamer and binds to the DNA where the DNA joins and leaves the octamer (see Figure 11.5). However, the results of recent experiments suggest that the H1 histone sits inside the coils of the nucleosome. Regardless of its position, H1 helps to lock the DNA into place, acting as a clamp around the nucleosome octamer.

Together, the core particle and its associated H1 his-tone are called the chromatosome, the next level of chro-matin organization. The H1 protein is attached to between 20 and 22 bp of DNA, and the nucleosome encompasses an additional 145 to 147 bp of DNA; so about 167 bp of DNA are held within the chromatosome. Chromatosomes are located at regular intervals along the DNA molecule and are separated from one another by linker DNA, which varies in size among cell types — most cells have from about 30 bp to 40 bp of linker DNA. Nonhistone chromosomal proteins may be associated with this linker DNA, and a few also appear to bind directly to the core particle.

Higher-order chromatin structure In chromosomes, adjacent nucleosomes are not separated by space equal to the length of the linker DNA; rather, nucleosomes fold on themselves to form a dense, tightly packed structure (see Figure 11.5). This structure is revealed when nuclei are gently broken open and their contents are examined with the use of an electron microscope; much of the chromatin that spills out appears as a fiber with a diameter of about 30 nm ( Figure 11.7a). A model of how this 30-nm fiber forms is shown in FIGURE 11.7b.

The next-higher level of chromatin structure is a series of loops of 30-nm fibers, each anchored at its base by proteins in the nuclear scaffold (see Figure 11.5). On average, each loop encompasses some 20,000 to 100,000 bp of

DNA and is about 300 nm in length, but the individual loops vary considerably. The 300-nm fibers are packed and folded to produce a 250-nm-wide fiber. Tight helical coiling of the 250-nm fiber, in turn, produces the structure that appears in metaphase: an individual chromatid approximately 700 nm in width.

(a) Core histones Linker DNA

of nucleosome /

(a) Core histones Linker DNA

of nucleosome /

11.6 The nucleosome is the fundamental repeating unit of chromatin. The space-filling model shows that the nucleosome core particle consists of two copies each of H2A, H2B, H3, and H4, around which DNA (white) coils. (Part d, from K. Luger et al., 1997, Nature 389:251; courtesy of T. H. Richmond.)

30-nm fiber

11.7 Adjacent nucleosomes pack together to form a 30-nm fiber. (Part a, Barbara Hamkalo, Molecular Biology and Biochemistry, University of California at Irvine.)

30-nm fiber

11.7 Adjacent nucleosomes pack together to form a 30-nm fiber. (Part a, Barbara Hamkalo, Molecular Biology and Biochemistry, University of California at Irvine.)

Concepts 9

The nucleosome consists of a core particle of eight histone proteins and DNA, about 146 bp in length, that wraps around the core. Chromato-somes, each including the core particle plus an H1 histone, are separated by linker DNA. Nucleosomes fold up to form a 30-nm chromatin fiber, which appears as a series of loops that pack to create a 250-nm-wide fiber. Helical coiling of the 250-nm fiber produces a 700-nm-wide chromatid.

www.whfreeman.com/pierce A virtual tour of nucleosome and chromatin structure and links to additional sites on chromatin structure and research

Changes in chromatin structure Although eukaryotic DNA must be tightly packed to fit into the cell nucleus, it must also periodically unwind to undergo transcription and replication. Evidence of the changing nature of chromatin structure is seen in the puffs of polytene chromosomes and in the sensitivity of genes to digestion by DNase I.

Polytene chromosomes are giant chromosomes found in certain tissues of Drosophila and some other organisms ( Figure 11.8). These large, unusual chromosomes arise when repeated rounds of DNA replication take place without accompanying cell divisions, producing thousands of copies of DNA that lie side by side. When polytene chromosomes are stained with dyes, numerous bands are revealed. Under certain conditions, the bands may exhibit chromosomal puffs — localized swellings of the chromosome. Each puff is a region of the chromatin that has relaxed its

11.8 Polytene chromosomes are giant chromosomes isolated from the salivary glands of larval Drosophila. Each puff represents a region of relaxed chromatin where transcription is taking place. (Andrew Syred/Science Photo Library/Photo Researchers.)

structure, assuming a more open state. If radioactively labeled uridine (a precursor to RNA) is briefly added to a Drosophila larva, radioactivity accumulates in chromosomal puffs, indicating that they are regions of active transcription. Additionally, the appearance of puffs at particular locations on the chromosome can be stimulated by exposure to hormones and other compounds that are known to induce the transcription of genes at those locations. This correlation between the occurrence of transcription and the relaxation of chromatin at a puff site indicates that chromatin structure undergoes dynamic change associated with gene activity.

A second piece of evidence indicating that chro-matin structure changes with gene activity is sensitivity to DNase I, an enzyme that digests DNA. The ability of this enzyme to digest DNA depends on chromatin structure: when DNA is tightly bound to histone proteins, it is less sensitive to DNase I, whereas unbound DNA is more sensitive to digestion by DNase I. The results of experiments that examine the effect of DNase I on specific genes show that DNase sensitivity is correlated with gene activity. For example, globin genes code for hemoglobin in the erythroblasts (precursors of red blood cells) of chickens. The forms of hemoglobin produced in chick embryos and chickens are different and are encoded by different genes ( FIGURE 11.9a). However, no hemoglobin is synthesized in chick embryos in the first 24 hours after fertilization. If DNase I is applied to chromatin from chick erythroblasts in this first 24-hour period, all the globin genes are insensitive to digestion ( FIGURE 11.9b). From day 2 to day 6 after fertilization, after hemoglobin synthesis has begun, the globin genes become sensitive to DNase I, and the genes that code for embryonic hemoglobin are the most sensitive ( FIGURE 11.9c). After 14 days of development, embryonic hemoglobin is replaced by the adult forms of hemoglobin. The most

Question: Is chromatin structure altered during transcription?

Method: Sensitivity to DNase I was tested on different tissues and at different times in development.

Key j DNA sensitive to DNase I [J Highest sensitivity to DNase I

Chicken DNA

Erythroblasts first 24 hours

Embryonic globin gene

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