Nucleosomes in metaphase chromosomes.

Previous studies of the structure of metaphase chromosomes have relied heavily on electron micrography and have revealed the existence of a 10-nm unit fiber that is thought to generate the native 23-30-nm fiber by higher order folding. The structural relationship of these metaphase fibers to the interphase fiber remains obscure. Recent studies on the digestion of interphase chromatin have revealed the existence of a regularly repeating subunit of DNA and histone, the nucleosome that generates the appearance of 10-nm beads connected by a short fiber of DNA seen on electron micrographs. It was therefore of interest to probe the structure of the metaphase chromosome for the presence of nucleosomal subunits. To this end metaphase chromosomes were prepared from colchicine-arrested cultures of mouse L-cells and were subjected to digestion with stayphylococcal nuclease. Comparison of the early and limit digestion products of metaphase chromosomes with those obtained from interphase nuclei indicates that although significant morphologic changes occur within the chromatin fiber during mitosis, the basic subunit structure of the chromatin fiber is retained by the mitotic chromosome.     

Intermediate structure between chromatin fibers and chromosome revealed by mechanical stretching and SPM measurement

The morphology of chromosomes (certain rod-shaped structures) is highly reproducible despite the high condensation of chromatin fibers (∼1 mm) into chromosomes (∼1 μm). However, the mechanism underlying the condensation of chromatin fibers into chromosomes is unclear. We assume that investigation of the internal structure of chromosomes will aid in elucidating the condensation process. In order to observe the detailed structure of a chromosome, we stretched a human chromosome by using a micromanipulator and observed its morphology along the stretched region by scanning probe microscopy (SPM). We found that the chromosome consisted of some fibers that were thicker than chromatin fibers. The found fiber was composed of approximately 90-nm-wide beads that were linked linearly. To explore the components of the fiber, we performed immunofluorescence staining of the stretched chromosome. Fluorescence signals of topoisomerase (Topo) IIα, which is known to interact with and support chromatin fibers, and DNA were detected both on the found fiber and beads. Furthermore, after micrococcal nuclease and trypsin treatments, the fibers were found to be mechanically supported by proteins. These results suggest that chromosome comprises an intermediate structure between chromatin fibers and chromosomes.     

Chromosome fibers studied by a spreading technique

Chromosomes and interphase nuclei can be spread on the surface of water in a simplified Langmuir trough. Interphase nuclei of Triturus erythrocytes display fibers with a diameter of about 250–300 Å. Very similar fibers are seen in metaphase chromosomes of cultured human cells. Fibers from grasshopper spermatocyte chromosomes (prophase) are more variable in diameter, and many fibers thinner than 200 Å extend laterally from the chromosome. In the grasshopper spermatocyte, fibers align in parallel to form plates. It is suggested that the 250–300 Å fibers may represent an inactive state of the chromosome material, and that only the thinner fibers are involved in RNA synthesis. The 250–300 Å fibers may result from the folding or coiling of a thinner fiber having the approximate dimensions of the nucleohistone molecule.     

Relationship between relative dry mass and average band width in regions of polytene chromosomes of Drosophila

Whole-mounted polytene chromosomes from Drosophila melanogaster were prepared for high-voltage electron microscopy. Relative dry mass of chromosome regions was estimated by densitometry of electron microscopic negatives. Comparison of dry mass of regions of the male X chromosome with that of regions of associated autosomes established that dry mass values are proportional to DNA content. Relative dry mass values of regions of polytene chromosomes from salivary glands, fat body, and malpighian tubules were correlated with the average diameter of bands in these regions: as mass doubled, band width increased by a factor of approximately 2. To provide a standard for estimating absolute levels of polyteny, band widths were measured for chromosomes representing one major polytene class, 256n. These chromosomes were observed to have an average band width of 0.9 m — These observations provide limits to models of chromatin organization in bands. For each chromatid, this area can accommodate up to five chromatin fibers of 250 Å diameter. This value may represent the extent of folding of a chromatin fiber in an average band. Alternatively, a chromatin fiber of higher-order structure could have a maximum diameter of 560 Å in an average band.     

DNA-protein binding in interphase chromosomes

The metachromatic dye, azure B, was analyzed by microspectrophotometry when bound to DNA fibers and DNA in nuclei with condensed and dispersed chromatin. The interaction of DNA and protein was inferred from the amount of metachromasy (increased β/α-peak) of azure B that resulted after specific removal of various protein fractions. Dye bound to DNA-histone fibers and frog liver nuclei fixed by freeze-methanol substitution shows orthochromatic, blue-green staining under specific staining conditions, while metachromasy (blue or purple color) results from staining DNA fibers without histone or tissue nuclei after protein removal. The dispersed chromatin of hepatocytes was compared to the condensed chromatin of erythrocytes to see whether there were differences in DNA-protein binding in "active" and "inactive" nuclei. Extraction of histones with 0.02 N HCl, acidified alcohol, perchloric acid, and trypsin digestion all resulted in increased dye binding. The amount of metachromasy varied, however; removal of "lysine-rich" histone (extractable with 0.02 N HCl) caused a blue color, and a purplish-red color (µ-peak absorption) resulted from prolonged trypsin digestion. In all cases, the condensed and the dispersed chromatin behaved in the same way, indicating the similarity of protein bound to DNA in condensed and dispersed chromatin. The results appear to indicate that "lysine-rich" histone is bound to adjacent anionic sites of a DNA molecule and that nonhistone protein is located between adjacent DNA molecules in both condensed and dispersed chromatin.     

THE HUMAN CHROMOSOME : Electron Microscopic Observations on Chromatin Fiber Organization

Human lymphocytes were grown in short-term tissue culture and were arrested in metaphase with Colcemid. Their chromosomes were prepared by the Langmuir trough-critical point drying technique and were examined under the electron microscope. In addition, some chromosomes were digested with trypsin, Pronase, or DNase. The chromosomes consist entirely of tightly packed, 240 ± 50-A chromatin fibers. Trypsin and Pronase treatments induce relaxation of fiber packing and reveal certain underlying fiber arrangements. Furthermore, trypsin treatment demonstrates that the chromatin fiber has a 25–50 A trypsin-resistant core surrounded by a trypsin-sensitive sheath. DNase digestion suggests that this core contains DNA.     

Chromosomal unit fibers in Drosophila

Chromosomal unit fibers consisting of long, regular fibers of about 0.40 m diameter were obtained from disintegrated, isolated chromosomes of two Drosophila melanogaster cell lines. In one cell line with an essentially normal karyotype, three clearly defined size classes of 15, 13, and 11 m length were observed corresponding to the three larger chromosomes of Drosophila. In a cell line carrying an additional translocation between the two largest chromosomes a 19 m fiber derived from the translocation chromosome was observed. Direct determinations of the DNA content per m length of Drosophila unit fibers show that DNA is contracted by a factor of about 1400x in agreement with calculations based on the length of the unit fibers and the known DNA content of the individual Drosophila chromosomes. These findings support our previously proposed model for the unit fiber sub-structure of chromosomes as being derived by a hierarchy of coiling with the corresponding contraction ratios being 7 (100 Å string of nucleosomes), 5 to 6 (250–300 Å thick nucleohistone fiber), and about 40 (unit fiber), resulting in a total contraction of DNA in unit fibers in the order of 1400x.     

A specific chromosome element,the telomere of Drosophila polytene chromosomes

The submicroscopic organization of terminal chromosome regions of Drosophila hydei polytene chromosomes is described. A compact region composed of tightly packed fibrils of 100 to 125 Å diameter embedded in an amorphous material is located at each of the chromosome ends of the 5 long chromosome arms. From this compact region, sometimes containing cavities, fibrils extend onto the nearest normal band region. The diameter of the extending fibrils is 100–125 Å, 200–250 Å or 400 Å. Pronase digestion of fixed and squashed chromosomes reduced the electron density of the amorphous matrix in the compact regions but failed to affect the diameter of the fibrils. The extending fibrils, however, showed a decrease in diameter after pronase digestion. The most frequently observed diameter values were 100–125 Å. — The volume of the terminal structures, including the compact region as well as the extending fibrils, is characteristically different for the various elements of the karyotype. Chromosome 2 displays the largest terminal structure, whereas chromosome 4 only occasionally shows the presence of compact regions. — End to end association of the long chromosome arms involves the fusion of the compact terminal structures. The non-random distribution of end to end association seems to be correlated with the volume of the terminal structures. Chromosome 2 which contains the largest compact terminal region is more frequently involved in end to end associations than any other chromosome arm. — The terminal regions show replication of DNA. They belong to the group of regions which display a discontinuous labeling pattern along the chromosomes, representing a late phase of the replication cycle. — The unique structural organization of the terminal chromosome regions, which is never observed at any other location of the genome supports the idea that they are morphological manifestations of the postulated telomeres.     

Higher order structure in metaphase chromosomes

The morphology of metaphase chromosome-derived chromatin fibers released from cells by non-ionic detergent cell lysis in the presence of divalent cations has been studied by electron microscopy. In these preparations the euchromatic arms appear as a series of loops, 200–300 Å in diameter, which are composed of closely-apposed nucleosome arrays. The higher order fiber in chromosomes derived from detergent-lysed cells appears to be less stable than chromatin fibers obtained by mechanical cell lysis. The fiber breaks down into a series of non-uniform nucleosome aggregates (superbeads) and finally to chromatin in a beads-on-a-string morphology upon incubation at 31° for 20 min. These observations allow us to suggest a relationship between uniform thick fibers, superbead-containing fibers, and beads-on-a-string chromatin within metaphase chromosomes.     

中华大蟾蜍染色体“单位线”结构的研究

适量BrdU处理中华大蟾蜍外周血淋巴细胞,常规制片后可观察到直径恒定为0.4μm的染色质纤维——单位线结构。本实验表明两栖类染色体同样具有单位线结构,并发现组成染色体两单体的姐妹单位线相伴排列,且与中期染色体有明显的形态联系。通过分析显示染色体和单位线过渡变化的分裂相,认为染色体高级结构由单位线进一步螺旋化完成,从而支持Bak模型。     

Comparison of human and mouse sperm chromatin structure by flow cytometry

Human and mouse sperm nuclei obtained by sonication or mechanical agitation of freshly isolated sperm in the presence of anionic detergent were purified through a sucrose gradient and stained with acridine orange (AO); their fluorescence intensity was measured by flow cytometry. The green fluorescence, characteristic of AO binding to DNA by intercalation, was twice lower per unit of DNA for human sperm nuclei than for human peripheral blood lymphocytes. After extraction of basic proteins with 0.08 N HCl, AO binding to DNA increased 3.2-fold for lymphocytes and only 1.3-fold for sperm indicating that, in contrast to somatic cells, the proteins restricting AO binding to DNA are essentially non-extractable from sperm at that low pH. Treatment of human and mouse nuclei with dithiothreitol (DTT), a sulfhydryl reducing agent, and trypsin, removed constraints responsible for the restriction of AO binding. Specifically, as a result of DTT treatment alone there was up to a 20–30% increase of AO binding; upon subsequent addition of trypsin there was a further rapid rise in AO binding up to a final level of approximately 5 times the original AO binding to isolated sperm nuclei. Electron microscopy of DTT-treated human sperm nuclei showed that the reducing agent caused chromatin decondensation to a level whereby 20–30 Å diameter fibers interconnecting chromatin bodies about 30–75 nm in diameter were revealed. Trypsin digestion in the presence of DTT converted the chromatin bodies into a network of fibrous structures about 150 Å in diameter. Both electron microscopy and flow cytometry demonstrated an extremely large intercellular variation among human sperm nuclei in response to DTT and trypsin treatment indicating heterogeneity of chromatin structure. In contrast, AO staining of mouse sperm nuclei increased homogeneously in response to DTT and trypsin treatment.     

The use of proteolytic enzymes to determine the location of histones in chromosomal substructures

We have observed that three proteolytic enzymes with widely different specificities produce a very similar pattern in terms of the order of digestion of the various histone fractions in chromatin. Histone H2A is most resistant to proteolytic attack by trypsin, chymotrypsin, or Pronase. H2B is next most resistant, followed by H3. Histone H1 is least resistant and is rapidly hydrolyzed by each of these enzymes. The behavior of histone H4 differs for the various enzymes. It is as resistant as H2A to digestion by trypsin and chymotrypsin but is readily hydrolyzed by Pronase. A comparison of the rates of digestion of the various histone fractions in chromatin with the rates in a DNA-free histone mixture and a study of the degradation products which result from digestion indicate that histone conformation and histone-histone and DNA-histone interactions are all involved in protecting histones from attack by proteolytic enzymes. From the results of our studies we have concluded that histones H1 and H3 are located in superficial positions of the chromosomal substructures (or nu bodies) while H2A is buried inside. Since histone H2B is relatively resistant to digestion but more readily degraded in chromatin than in a DNA-free histone mixture, it is difficult to determine its chromosomal location. Histone H4 behaves as if a large portion of the molecule is located in the major groove of the DNA helix.     

Packing of the 30 nm chromatin fiber in the human metaphase chromosome

The human genetic material is packed hierarchically within the metaphase chromosome: the DNA moleculet together with histone proteins form 11 nm diameter nucleosomes, which are then ordered into the 30 nm thick chromatin fiber. Little is known about the packing of this fiber within the chromosome. We have developed a tracking algorithm with which we followed its path within a three-dimensional reconstruction of a human chromosome computed from a series of electron micrographie projections. Fiber segments were seen to form loops of 100–350 nm diameter. Our observations indicate that these loops — which themselves show no preferred orientation — are organised into regions of roughly 200 nm axial extent.     

Chromatin elimination in the hypotrichous ciliate Stylonychia mytilus

Chromatin elimination in the hypotrichous ciliate Stylonychia mytilus was studied by means of electron microscopy using a microspreading procedure. In the polytene chromosomes of the macronuclear anlagen three organization patterns are observed: Bands of various size composed of 300 Å chromatin fibers, large blocks of 300 Å nucleofilaments which probably represent the heterochromatic regions of the chromosome and axial 120 Å filaments. Those DNA sequences which become eliminated belong to the 300 Å fiber type. The eliminated chromatin occurs in the form of rings of variable size corresponding to a DNA content between 18 and 160 Kb while the axial 120 Å filaments appear to be preserved.     

Effects of tethering HP1 to euchromatic regions of the Drosophila genome

Heterochromatin protein 1 (HP1) is a conserved non-histone chromosomal protein enriched in heterochromatin. On Drosophila polytene chromosomes, HP1 localizes to centric and telomeric regions, along the fourth chromosome, and to specific sites within euchromatin. HP1 associates with centric regions through an interaction with methylated lysine nine of histone H3, a modification generated by the histone methyltransferase SU(VAR)3-9. This association correlates with a closed chromatin configuration and silencing of euchromatic genes positioned near heterochromatin. To determine whether HP1 is sufficient to nucleate the formation of silent chromatin at non-centric locations, HP1 was tethered to sites within euchromatic regions of Drosophila chromosomes. At 25 out of 26 sites tested, tethered HP1 caused silencing of a nearby reporter gene. The site that did not support silencing was upstream of an active gene, suggesting that the local chromatin environment did not support the formation of silent chromatin. Silencing correlated with the formation of ectopic fibers between the site of tethered HP1 and other chromosomal sites, some containing HP1. The ability of HP1 to bring distant chromosomal sites into proximity with each other suggests a mechanism for chromatin packaging. Silencing was not dependent on SU(VAR)3-9 dosage, suggesting a bypass of the requirement for histone methylation.     

Contributions of linker histones and histone H3 to chromatin structure: scanning force microscopy studies on trypsinized fibers.

Little is known about the mechanisms that organize linear arrays of nucleosomes into the three-dimensional structures of extended and condensed chromatin fibers. We have earlier defined, from scanning force microscopy (SFM) and mathematical modeling, a set of simple structural determinants of extended fiber morphology, the critical parameters being the entry-exit angle between consecutive linkers and linker length. Here we study the contributions of the structural domains of the linker histones (LHs) and of the N-terminus of histone H3 to extended fiber morphology by SFM imaging of progressively trypsinized chromatin fibers. We find that cleavage of LH tails is associated with a lengthening of the internucleosomal center-to-center distance, and that the somewhat later cleavage of the N-terminus of histone H3 is associated with a flattening of the fiber. The persistence of the "zigzag" fiber morphology, even at the latest stages of trypsin digestion, can be attributed to the retention of the globular domain of LH in the fiber.     

Linker histone H1 per se can induce three-dimensional folding of chromatin fiber

Higher-order architectures of chromosomes play important roles in the regulation of genome functions. To understand the molecular mechanism of genome packing, an in vitro chromatin reconstitution method and a single-molecule imaging technique (atomic force microscopy) were combined. In 50 mM NaCl, well-stretched beads-on-a-string chromatin fiber was observed. However, in 100 mM NaCl, salt-induced interaction between nucleosomes caused partial aggregation. Addition of histone H1 promoted a further folding of the fiber into thicker fibers 20-30 nm in width. Micrococcal nuclease digestion of these thicker fibers produced an approximately 170 bp fragment of nucleosomal DNA, which was approximately 20 bp longer than in the absence of histone H1 ( approximately 150 bp), indicating that H1 is correctly placed at the linker region. The width of the fiber depended on the ionic strength. Widths of 20 nm in 50 mM NaCl became 30 nm as the ionic strength was changed to 100 mM. On the basis of these results, a flexible model of chromatin fiber formation was proposed, where the mode of the fiber compaction changes depending both on salt environment and linker histone H1. The biological significance of this property of the chromatin architecture will be apparent in the closed segments ( approximately 100 kb) between SAR/MAR regions.     

A repeating unit of higher order chromatin structure in chick red blood cell nuclei

The organization of nucleosomes in higher order chromatin structures has been studied by electron microscopy of chick red blood cell nuclei. Chromatin appears as a thick fiber with an average diameter of approximately 300 Å when prepared for electron microscopy in buffers which approximate physiological ionic strength. Progressive steps of disassembly of the thick fiber into individual nucleosomes could be induced either by ionic strength reduction or by tRNA treatment (which removes histone H1 and some non-histone chromosomal proteins). When disassembly was induced by ionic strength reduction in the presence of Mg⁺⁺ (or Ca⁺⁺), the lengths of the intermediate disassembly products were found to be multiples of 330 Å. The diameter of these structures was estimated to be 275 Å. This intermediate in the disassembly process is not observed if thick fiber disassembly is induced by ionic strength reduction in the absence of divalent cations. To investigate whether the higher order structural unit is present in the thick fiber at physiological ionic strengths, tRNA treatment was used to induce thick fiber disassembly under physiological monovalent ionic conditions. In this case, either with or without divalent cations, a supranucleosomal unit was found with dimensions similar to those given above. This data provides evidence for a slightly oblong supranucleosomal structure (330 × 275 Å) which forms a repeating unit in the chromatin thick fiber.     

Native and reconstituted chromosome fiber fragments

The structure of hen erythrocyte chromatin fibers was studied with the electron microscope. Chromatin fiber fragments with a length of about 5,000 Å and an average diameter of 320 Å are composed of 13 globular subunits (superbeads) which contain different numbers of nucleosomes. Their number average corresponds to 17 nucleosomes. — The interaction of lysine-rich histones with nucleosome chains was investigated by reconstitution experiments and was found to be semi-cooperative.     

The mechanism and pattern of banding induced by restriction endonucleases in human chromosomes

The mechanism of chromosome banding induced by restriction endonucleases was analyzed by measuring the amount of radioactivity extracted from [¹⁴C]thymidine-labeled chromosomes digested first with restriction enzymes and subsequently with proteinase K and DNase I. Restriction enzymes with a high frequency of recognition sites in the DNA produced a large number of short DNA fragments, which were extracted from chromosomes during incubation with the enzyme. This loss of DNA resulted in decreased chromosomal staining, which did not occur in regions resistant to restriction enzyme digestion and thus led to banding. Subsequent digestion of chromosomes with proteinase K produced a further loss of DNA, which probably corresponded to long fragments retained in the chromosome by the proteins of fixed chromatin. Restriction enzymes induce chromatin digestion and banding in G1 and metaphase chromosomes, and they induce digestion and the appearance of chromocenters in interphase nuclei. This suggests that the spatial organization and folding of the chromatin fibril plays little or no role in the mechanism of chromosome banding.It was confirmed that the pattern of chromosome banding induced by AluI, MboI, HaeIII, DdeI, RsaI, and HinfI is characteristic for each endonuclease. Moreover, several restriction banding polymorphisms that were not found by conventional C-banding were detected, indicating that there may be a range of variability in the frequency and distribution of restriction sites in homologous chromosome regions.