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本课件为《分子生物学》第19章核小体（Nucleosomes），依据 Gene VII 教材编排，沿Prions cause、The nucleosome等小节推进，讲解本章核心概念与分子机制，配逐页图示与文字版。
Chapter 19 Nucleosomes
Figure 18.9 The sister chromatids of a mitotic pair each consist of a fiber (~30 nm in diameter) compactly folded into the chromosome. Photograph kindly provided by E. J. Du Praw.
Micrococcal nuclease is an endonuclease that cleaves DNA; in chromatin, DNA is cleaved preferentially between nucleosomes.
Figure 19.1 Chromatin spilling out of lysed nuclei consists of a compactly organized series of particles. The bar is 100 nm. Photograph kindly provided by Pierre Chambon.
Figure 19.2 Individual nucleosomes are released by digestion of chromatin with micrococcal nuclease. The bar is 100 nm. Photograph kindly provided by Pierre Chambon.
Figure 19.3 The nucleosome consists of approximately equal masses of DNA and histones (including H1). The predicted mass of the nucleosome is 262 kD.
Figure 19.4 The nucleosome may be a cylinder with DNA organized into two turns around the surface.
Figure 19.5 The two turns of DNA on the nucleosome lie close together.
Figure 19.6 Sequences on the DNA that lie on different turns around the nucleosome may be close together.
Figure 19.7 Micrococcal nuclease digests chromatin in nuclei into a multimeric series of DNA bands that can be separated by gel electrophoresis. Photograph kindly provided by Markus Noll.
Figure 19.8 Each multimer of nucleosomes contains the appropriate number of unit lengths of DNA. Photograph kindly provided by John Finch.
Figure 19.9 Micrococcal nuclease reduces the length of nucleosome monomers in discrete steps. Photograph kindly provided by Roger Kornberg.
Figure 19.10 Microccocal nuclease initially cleaves between nucleosomes. Mononucleosomes typically have ~200 bp DNA. End-trimming reduces the length of DNA first to ~165 bp, and then generates core particles with 146 bp.
Figure 19.4 The nucleosome may be a cylinder with DNA organized into two turns around the surface.
Figure 19.11 Nicks in double-stranded DNA are reve a led by fragments when the DNA is denatured to give single strands. If the DNA is labeled at (say) 5ends, only the 5fragments are visible by autoradiography. The size of the fragment identifies the distance of the nick from the labeled end.
Figure 2.4 When restriction fragments are identified by their possession of a labeled end, each fragment directly shows the distance of a cutting site from the end. Successive fragments increase in length by the distance between adjacent restriction sites.
Figure 19.12 Sites for nicking lie at regular intervals along core DNA, as seen in a DNAase I digest of nuclei. Photograph kindly provided by Leonard Lutter.
Figure 19.13 Two num be ring schemes divide core particle DNA into 10 bp segments. Sites may be num be red S1 to S13 from one end; or taking S7 to identify coordinate 0 of the dyad symmetry, they may be num be red -7 to +7.
Figure 19.4 The nucleosome may be a cylinder with DNA organized into two turns around the surface.
Figure 19.14 The most exposed positions on DNA recur with a periodicity that reflects the structure of the double helix. (For clarity, sites are shown for only one strand.).
Figure 19.15 High resolution analysis shows that each site for DNAase I consists of several adjacent susceptible phosphodiester bonds as seen in this example of sites S4 and S5 analyzed in end-la be led core particles. Photograph kindly provided by Leonard Lutter.
Figure 19.16 The supercoils of the SV40 minichromosome can be relaxed to generate a circular structure, whose loss of histones then generates supercoils in the free DNA.
Figure 19.4 The nucleosome may be a cylinder with DNA organized into two turns around the surface.
Figure 19.17 The 10 nm fiber in partially unwound state can be seen to consist of a string of nucleosomes. Photograph kindly provided by Barbara Hamkalo.
Figure 19.18 The 10 nm fiber is a continuous strong of nucleosomes.
Figure 19.19 The 30 nm fiber has a coiled structure. Photograph kindly provided by Barbara Hamkalo.
Figure 19.20 The 30 nm fiber may have a helical coil of 6 nucleosomes per turn, organized radially.
Figure 19.21 In a symmetrical model for the nucleosome, the H32-H42 tetramer provides a kernel for the shape. One H2A-H2B dimer can be seen in the top view; the other is underneath.
Figure 19.22 The crystal structure of the histone core octamer is represented in a space-filling model with the H32-H42 tetramer shown in white and the H2A-H2B dimers shown in blue. Only one of the H2A-H2B dimers is visible in the top view, because the other is hidden underneath. The potential path of the DNA is shown in the top view as a narrow tube (one quarter the diameter of DNA), and in the side view by the parallel lines in a 20 ? wide bundle. Photographs kindly provided by Evangelos Moudrianak is.
Figure 19.4 The nucleosome may be a cylinder with DNA organized into two turns around the surface.
Figure 19.23 Histone positions in a top view show H3-H4 and H2A-H2B pairs in a half nucleosome; the symmetrical organization can be seen in the superimpositi on of both halves.
Figure 19.24 The globular bodies of the histones are localized in the histone octamer of the core particle, but the locations of the N-terminal tails, which carry the sites for modification, are not known, and could be more flexible.
Figure 19.25 Acetyl at ion of lysine or phosphorylation of serine reduces the overall positive charge of a protein.
Figure 19.26 Replicated DNA is immediately incorporated into nucleosomes. Photograph kindly provided by S. Mac Knight.
Figure 19.27 In vitro, DNA can either interact directly with an intact (crosslinked) histone octamer or can assemble with the H32-H42 tetramer, after which two H2A-H2B dimers are added.
Figure 19.28 If histone octamers were conserved, old and new octamers would band at different densities when replication of heavy octamers occurs in light amino acids (part 1); but actually the octamers band diffusely between heavy and light densities, suggesting disassembly and reassembly (part 2).
Figure 19.29 Nucleosome positioning places restriction sites at unique positions relative to the linker sites cleaved by micrococcal nuclease.19.9 Do nucleosomes lie at specific positions?.
Figure 19.30 In the absence of nucleosome positioning, a restriction site lies at all possible locations in different copies of the genome. Fragments of all possible sizes are produced when a restriction enzyme cuts at a target site (red) and micrococcal nuclease cuts at the junctions between nucleosomes (green).19.9 Do nucleosomes lie at specific positions?.
Figure 19.31 Translational positioning describes the linear position of DNA relative to the histone octamer. Displacement of the DNA by 10 bp changes the sequences that are in the more exposed linker regions, but does not alter which face of DNA is protected by the histone surface and which is exposed to the exterior. DNA is really coiled around the nucleosomes, and is shown in linear form only for convenience.19.9 Do nucleosomes lie at specific positions?.
Figure 19.32 Rotational positioning describes the exposure of DNA on the surface of the nucleosome. Any movement that differs from the helical repeat (~10.2 bp/turn) displaces DNA with reference to the histone surface. Nucleotides on the inside are more protected against nucleases than nucleotides on the out side.19.9 Do nucleosomes lie at specific positions?.
Figure 19.33 The extended axis of an rDNA transcription unit alternates with the only slightly less extended non-transcribed spacer. Photograph kindly provided by Charles Laird.19.10 Are transcribed genes organized in nucleosomes?.
Figure 19.34 An SV40 minichromosome can be transcribed. Photograph kindly provided by Pierre Chambon.19.10 Are transcribed genes organized in nucleosomes?.
Figure 19.35 RNA polymerase is comparable in size to the nucleosome and might encounter difficulties in following the DNA around the histone octamer.19.10 Are transcribed genes organized in nucleosomes?.
Figure 19.36 A protocol to test the effect of transcription on nucleosomes shows that the histone octamer is displaced from DNA and rebinds at a new position.19.10 Are transcribed genes organized in nucleosomes?.
Figure 19.37 RNA polymerase displaces DNA from the histone octamer as it advances. The DNA loops back and attaches (to polymerase or to the octamer) to form a closed loop. As the polymerase proceeds, it generates positive supercoiling ahead. This displaces the octamer, which keeps contact with DNA and/or polymerase, and is inserted behind the RNA polymerase.19.10 Are transcribed genes organized in nucleosomes?.
Figure 19.38 The URA3 gene has translationally positioned nucleosomes before transcription. When transcription is induced, nucleosome positions are randomized. When transcription is repressed, the nucleosomes resume their particular positions. Photograph kindly provided by Fritz Thoma.19.10 Are transcribed genes organized in nucleosomes?.
Figure 19.39 Indirect end-labeling identifies the distance of a DNAase hypersensitive site from a restriction cleavage site. The existence of a particular cutting site for DNAase I generates a discrete fragment, whose size indicates the distance of the DNAase I hypersensitive site from the restriction site.
Figure 19.40 The SV40 minichromosome has a nucleosome gap. Photograph kindly provided by Moshe Yaniv.
Figure 19.41 The SV40 gap includes hypersensitive sites, sensitive regions, and a protected region of DNA. The hypersensitive site of a chicken b-globin gene comprises a region that is susceptible to several nucleases.
Domain of a chromosome may refer either to a discrete structural entity defined as a region within which supercoiling is independent of other domains; or to an extensive region including an expressed gene that has heightened sensitivity to degradation by the enzyme DNAase I.
Figure 19.42 Sensitivity to DNAase I can be measured by determining the rate of disappearance of the material hybridizing with a particular probe.
Figure 19.43 In adult erythroid cells, the adult b-globin gene is highly sensitive to DNAase I digestion, the embryonic b-globin gene is partially sensitive (probably due t In adult erythroid cells, the adult b-globin gene is highly sensitive to DNAase I digestion, the embryonic b-globin gene is partially sensitive (probably due to spreading effects), but ovalbum in is not sensitive. Data kindly provided by Harold Weintraub.
Epigenetic changes influence the phenotype without altering the genotype. They consist of changes in the properties of a cell that are inherited but that do not represent a change in genetic information.
Figure 19.44 Position effect varieg at ion in eye color results when the white gene is integrated near heterochromatin. Cells in which white is inactive give patches of white eye, while cells in which white is active give red pa Position effect varieg at ion in eye color results when the white gene is integrated near heterochromatin. Cells in which white is inactive give patches of white eye, while cells in which white is active give red patches. The severity of the effect is determined by the closeness of the integrated gene to heterochromatin. Photograph kindly provided by Steve Henikoff.
Figure 19.45 Extension of heterochromatin inactivates genes. The probability that a gene will be inactivated depends on its distance from the heterochromatin region.
Figure 19.46 Formation of heterochromatin is initiated when RAP1 binds to DNA. SIR3/4 bind to RAP1 and also to histones H3/H4. The complex polymerizes along chromatin and may connect telomeres to the nuclear matrix.
Figure 19.47 Different means of dosage compens at ion are used to equalize X chromosome expression in male and female.
Figure 19.48 X-linked varieg at ion is caused by the random inactiv at ion of one X chromosome in each precursor cell. Cells in which the + allele is on the active chromosome have wild phenotype; but cells in which the -allele is on the active chromosome have mutant phenotype.
Figure 19.49 X-inactiv at ion involves stabiliz at ion of XIST RNA, which coats the inactive chromosome.
Imprinting describes a change in a gene that occurs during passage through the sperm or egg with the result that the paternal and maternal alleles have different properties in the very early embryo. May be caused by methylation of DNA.
Figure 19.50 The state of methylated sites could be perpetuated by an enzyme that recognizes only hemimethylated sites as substrates.
Figure 19.51 The state of methylation is controlled by three enzymes.
Figure 19.52 The parental alleles of Igf2 are differentially methylated in the early embryo, but the patterns of methylation are reset when gametes are formed by the adult.
Prion is a proteinaceous infectious agent, which behaves as an inheritable trait, although it contains no nucleic acid. Examples are PrPSc, the agent of scrapie in sheep and bovine spongiform encephalopathy, and Psi, which confers an inherited state in yeast.
Figure 19.50 The state of methylated sites could be perpetuated by an enzyme that recognizes only hemimethylated sites as substrates.
Figure 19.53 What happens to protein complexes on chromatin during replication?.
Figure 19.45 Extension of heterochromatin inactivates genes. The probability that a gene will be inactivated depends on its distance from the heterochromatin region.
Figure 19.54 Acetylated cores are conserved and distributed at random to the daughter chromatin fibers at replication. Each daughter fiber has a mixture of old (acetylated) cores and new (unacetylated) cores.
Figure 19.55 The state of the Sup35 protein determines whether termination of translation occurs.
Figure 19.56 Newly synthesized Sup35 protein is converted into the [PSI+] state by the presence of pre-existing [PSI+] protein.
Figure 19.57 Purified protein can convert the[psi-] state of yeast to [PSI+].
Scrapie is a infective agent made of protein.
Figure 19.58 A Prp Sc protein can only infect an animal that has the same type of endogenous PrPC protein.
1.All eukaryotic chromatin consists of nucleosomes.2.The path of DNA around the histone octamer creates3.Nucleosomes are organized into a fiber of 30 nm diameter which has 6 nucleosomes per turn and a packing ratio of 40. 4.RNA polymerase displaces histone octamers during transcription. 5.Two types of changes in sensitivity to nucleases are associated with gene activity. 6.Formation of heterochromatin may be initiated at certain sites and then propagated for a distance that is not precisely determined.
7.Inactive chromatin at yeast telomeres and silent mating type loci appears to have a common cause, and involves the interaction of certain proteins with the N-terminal tails of histones H3 and H4.8.Inactiv at ion of one X chromosome in female (eutheri an) mammals occurs at random. 9. Methylation of DNA is inherited epigenetically.10.Prions are proteinaceous infectious agents that are responsible for the disease ofscrapie in sheep and for related diseases in man.
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