Chromonema and chromomere

A study of ultrathin sections of normal Chinese hamster cells and cells treated with decreasing concentrations of bivalent cations (Ca²⁺ and Mg²⁺) in situ revealed several discrete levels of compaction of DNA-nucleoprotein (DNP) fibrils in mitotic chromosomes and the chromatin of interphase nuclei. At concentrations ranging from 3 mM CaCl₂ and 1 mM MgCl₂ to ten times less, the chromosomes are found to contain fibrous elements (chromonemata) about 100 nm in diameter. As Ca²⁺ concentration is gradually decreased to 0.2–0.1 mM, the chromosomes decondense into a number of discrete chromatin structures, the chromomeres. As decondensation proceeds, these chromomeres acquire a rosettelike structure with DNP fibrils radiating from an electron-dense core. Upon complete decondensation of chromosomes, individual chromomeres persist only in the centromeric regions. The following levels of DNP compaction in mitotic chromosomes are suggested: a 10-nm nucleosomal fibril, a 25-nm nucleomeric fibril, and the chromonema, a fibrous structure, about 100 nm in diameter, composed of chromomeres. Interphase nuclei also contain structures which are morphologically similar to the chromomeres of mitotic chromosomes.     

Chromatin fibers observed in situ in frozen hydrated sections. Native fiber diameter is not correlated with nucleosome repeat length

Chromatin fibers have been observed and measured in frozen hydrated sections of three types of cell (chicken erythrocytes and sperm of Patiria miniata and Thyone briareus) representing an approximately 20- bp range of nucleosomal repeat lengths. For sperm of the starfish P. miniata, it was possible to obtain images of chromatin fibers from cells that were swimming in seawater up to the moment of cryo- immobilization, thus providing a record of the native morphology of the chromatin of these cells. Glutaraldehyde fixation produced no significant changes in the ultrastructure or diameter of chromatin fibers, and fiber diameters observed in cryosections were similar to those recorded after low temperature embedding in Lowicryl K11M. Chromatin fiber diameters measured from cryosections of the three types of nuclei were similar, a striking contrast to the situation for chromatin isolated from these cell types, where a strong positive correlation between diameter and nucleosomal repeat length has been established. The demonstration of chromatin fibers in unfixed whole cells establishes an unequivocal baseline for the study of native chromatin and chromosome architecture. The significant differences between chromatin fibers in nucleo and after isolation supports a previous observation (P. J. Giannasca, R. A. Horowitz, and C. L. Woodcock. 1993. J. Cell Sci. 105:551-561), and suggests that structural studies on isolated material should be interpreted with caution until the changes that accompany chromatin isolation are understood.     

Ultrastructure of nuclei isolated from plant protoplasts

Summary A simple method, involving selective Triton X-100 membrane solubilization, has been developed for the isolation of nuclei from barley and tobacco protoplasts which gives a high yield of essentially pure nuclei. The isolated nuclei resembled those in leaf cells and protoplasts when the isolated nuclei were fixed for short times (2 hours, Medium II), except that their chromatin appeared to be more highly condensed and barley nuclei also lacked the outer nuclear membrane. When longer times of fixation (12 hours, Medium I) were used, the isolated nuclei lacked the characteristic condensed chromatin appearance.     

An electron microscopic study of mitosis in mouse duodenal crypt cells confirms that the prophasic condensation of chromatin begins during the DNA-synthesizing (S) stage of the cycle

The phases of mitosis were examined in the columnar cells at the base of duodenal crypts in adult male mice given an intravenous injection of 3H-thymidine and sacrificed 20 min later. The duodenum was fixed by immersion into glutaraldehyde-formaldehyde, and the cells were examined in the electron microscope, with or without processing for radioautography. Interphase nuclei are characterized by the distribution of chromatin; aside from the cortical chromatin spread along nuclear envelope and nucleolus, there are chromatin accumulations that belong mainly in two different classes: 1) numerous chromatin "specks" ranging in size from about 5 to 70 nm and averaging 47 nm; 2) a few roughly circular or elongated chromatin "packets" measuring from 70 to 230 nm. Early prophase nuclei differ mainly by a large increase in the number of chromatin packets to 20-30 or more per nuclear profile; their average diameter is 128 nm. During mid-prophase, the chromatin packets enlarge gradually to an average 221 nm diameter. Between mid- and late prophase, there is a further increase in diameter to 679 nm. At metaphase, the packets take on the appearance of mature chromosomes, and their diameter increases to 767 nm. At anaphase, daughter chromosomes migrate to each pole, where they fuse into a compact chromatin mass. At telophase, nucleoplasmic areas progressively enlarge within the chromatin mass and separate strands of chromatin, which gradually become segmented into chromatin clumps. Counts of mitotic cells show a high proportion of prophase and telophase nuclei. Calculation from the counts yields the duration of the phases, that is, 5.6, 0.2, 0.1, and 1.6 hr, respectively, for pro-, meta-, ana-, and telophase. Finally, radioautography 20 min after 3H-thymidine injection shows labeling in 54% of the interphase nuclei, 85% of early prophase nuclei, and 73% of mid-prophase nuclei, while there is no label in late prophase, metaphase, anaphase and telophase nuclei. In confirmation of previous light microscopic work, the S stage of the cycle begins when a cell is in interphase and continues through the early prophase and part of mid-prophase. Moreover, the main sites of DNA synthesis are the chromatin specks during interphase and the cortical chromatin during early and mid-prophase. The chromosome condensation taking place in the meantime may be separated into two main steps: 1) a slow, moderate condensation of the chromatin packets during early and mid-prophase and 2) a rapid, pronounced one during late prophase and prometaphase when the packets become chromosomes.     

Electron microscopic and biochemical evidence that chromatin structure is a repeating unit

Electron microscopic and biochemical studies demonstrate that the fundamental structure of chromatin depleted of lysine-rich histones is composed of a flexible chain of spherical particles (nucleosomes), about 125 Å in diameter, connected by DNA filaments. Such a chromatin preparation can be separated by centrifugation into two fractions which differ in the spacing of the nucleosomes. In one fraction almost all of the DNA is condensed in nucleosomes, while the other fraction contains long stretches of free DNA connecting regions where the nucleosomes are closely packed. The isolated nucleosomes contain about 200 base pairs of DNA and the four histones F2a₁, F2a₂, and F2b, and F3 in an overall histone/DNA ratio of 0.97. In such a structure the DNA is compacted slightly more than five times from its extended length. The same basic structure can be visualized in chromatin spilling out of lysed nuclei. However, in this latter case the nucleosomes are very closely packed, suggesting that histone F1 is involved in the superpacking of DNA in chromosomes and nuclei. The chromatin fiber appears to be a self-assembling structure, since the nucleosomal arrangement can be reconstituted in vitro from DNA and the four histones F2a₁, F2a₂, F2b and F3 only, irrespective of their cellular origin.     

Ultrastructure of the DNP fibrils and of the interchromatin granules in isolated nuclei of the rat liver]

The structural organization of DNP fibrils and interchromatin granules of isolated rat hepatocyte nuclei has been studied in various conditions of chromatin solubilization. When observed either in nuclei fixed in situ or in a solution containing 20 mM TEA and 1 mM MgCl2, a DNP fibril consists of globular structures 20--25 nm in diameter. In the nuclei fixed in a magnesium-free solution (20 mM TA), nucleosome structures are revealed in DNP. Condensation of chromatin results from interaction between 20 nm globular fibrils, whereas the complete dispersion of chromatin is a consequence of its conversion into the nucleosomal form. In the conditions of both DNP structuralization and dispersion, the nuclei are revealed to contain zones of interchromatin granules connected by thin fibrils. It is assumed that the different compactness of these granular-fibrillar complexes and of the regions of condensed chromatin may be used for their separation and fractionation.     

Structural repeating units in chromatin: I. Evidence for their general occurrence

When chromatin from a diverse range of eukaryotes is prepared for the electron microscope by aldehyde fixation followed by centrifugation onto the grid, the fibers are composed of interconnected spherical particles, about 70 Å in diameter. Evidence is presented which suggests that the particles are an in vivo structural repeating unit of deoxyribonucleoprotein, and not a preparation-induced artefact.     

Digestion by micrococcal nuclease of mouse submandibular-salivary-gland chromatin.

Nucleic prepared from mouse submandibular salivary gland show marked fragility during isolation. Hwever, intact nuclei relatively free from cytoplasmic contamination were obtained by homogenization in buffers containing 0.88 M-sucrose, Ca2+, spermine, spermidine and the proteinase inhibitor aprotinin, followed by centrifugation through 2.2 M-sucrose. The kinetics of digestion by the micrococcal nuclease of chromatin in these nuclei are similar to those of chromatin from mouse liver nuclei. Base-pair size analysis of the solubilized DNA from both organs shows a stable high-molecular weight species of chromatin, which is further digested to mononucleosome and subnucleosome species. With extensive digestion the chromatin becomes insoluble. The mononucleosomes produced from salivary-gland chromatin after the inhibition of endogenous proteinase activity exhibit an s20,w value of 11S and contain histones H1, H2A, H2B, H3 and H4.     

DNAase I and cellular factors that affect chromatin structure

The use of DNAase I as a probe of chromatin structure is frequently fraught with problems of irreproducibility. We have recently evaluated this procedure, documented the sources of the problems, and standardized the method for reproducible results (Prentice and Gurley (1983) Biochim. Biophys. Acta 740, 134–144). We have now used this probe to detect differences in chromatin structure between cells blocked (1) in G₁ phase by isoleucine deprivation, or (2) in early S phase by sequential use of isoleucine deprivation followed by release into the presence of hydroxyurea. The cells blocked in G₁ phase have easily-digestible chromatin, while cells blocked in early S phase have chromatin which is much more resistant to DNAase I. These differences were found to be the result of diffusible factors found in the cytoplasm and nuclei of G₁- and S-phase cells, respectively. The G₁ cells contained a cytoplasmic factor which modulates the chromatin structure of S-phase nuclei to a more easily digestible state, while cells blocked in S phase contain a nuclear factor which modulates the chromatin structure of G₁ nuclei to a state more resistant to digestion. DNAase I is much more sensitive to these cell cycle-specific chromatin changes than is micrococcal nuclease. The results indicate that, under controlled conditions, DNAase I should be a valuable probe for detecting chromatin structural changes associated with cell cycle traverse, differentiation, development, hormone action and chemical toxicity.     

Ultrastructure of meristematic cells of dormant and released buds in Tradescantia paludosa

The lateral bud meristems of Tradescantia paludosa show a characteristic cytohistological zonation during dormancy. The cells comprising this so called ‘zone of inhibition’, which is located at the extreme tip of the bud apex, rarely synthesize nuclear DNA or undergo mitotic division. These nuclei are as large as prophase nuclei, yet contain only telophase (2C) amounts of DNA and significantly lower amounts of histone as compared to the 2C nuclei of the actively dividing cells.Ultrastructural observations of the nuclei in the ‘zone of inhibition’ show that a large proportion of the chromatin is organized as less condensed, diffuse, euchromatin fibrils; however, the chromatin of the actively dividing nuclei of the cells outside the ‘zone of inhibition’ or in the released bud meristems is organized to a greater extent as condensed clumps of heterochromatin. When the dormancy is released, the nuclei in the ‘zone of inhibition’ synthesize DNA and histone and undergo cell division in approx. 4 days. Striking changes in the organization of chromatin fibrils take place during this transition period. The diffuse chromatin fibrils of the nuclei in the ‘zone of inhibition’ progressively become more and more condensed as the cell prepares to undergo the first mitotic division after the release of dormancy. This change which is coupled with the synthesis of histones in the nuclei of the ‘zone of inhibition’ suggests a prominent structural role of these basic proteins in the organization of the chromatin. The large volume of 2C nuclei of the ‘zone of inhibition’ seems, therefore, to result not from a great nuclear mass, but probably from a relatively small degree of condensation of chromatin.     

Ultrastructural changes in vitro of rat liver nucleoli in response to polyamines

Summary Structural alterations of the nucleoli of rat liver cells were noted when these nuclei were isolated with spermidine or spermine rather than magnesium. When 5–10 mM spermidine or spermine were used to isolate the nuclei, the nucleoli were a) larger, b) contained numerous and sometimes large lacunae, and c) were less aggregated and had prominent chromatin caps. These chromatin caps gave the nucleolus a ring-shaped appearance in the light microscope. These findings, coupled with physiological data that indicate that polyamines enhance nucleolar RNA polymerase activity (Russell et al., 1971), suggest that spermidine and spermine may be involved in the control of ribosomal RNA synthesis. To our knowledge, this is the first instance of the direct stimulation of ribosomal RNA synthesis during nuclear isolation.Supported by USPHS Grant NS-07934.     

RNA TRANSPORT FROM NUCLEUS TO CYTOPLASM IN CHIRONOMUS SALIVARY GLANDS

The fine structure and cytochemistry of the extremely large RNA puffs, or Balbiani rings, in salivary gland nuclei of midge, Chironomus thummi, larvae have been investigated. The Balbiani rings are composed of a diffuse mass of electron-opaque 400 to 500 A granules, short threads about 180 to 220 A in diameter and associated fine chromatin fibrils. These components appear to be organized into brushlike elements which form the ring. Electron microscope cytochemistry has shown that the granules and short threads contain RNA. After ribonuclease digestion, only 50 to 100 A chromatin fibrils were apparent in the Balbiani ring, and the granules were no longer demonstrable. Deoxyribonuclease digestion had no apparent effect on these structures. Observations indicate that the granules are formed from the short threads and released into the nucleoplasm in which they are evenly distributed. At the nuclear envelope, many granules have been observed partially or completely within the nuclear pores. These granules become elongated and are shown to penetrate the center of the pore in a rodlike form, about 200 A in diameter. The Balbiani ring granules are not normally visible within the cytoplasm adjacent to the nuclear envelope, but have been rarely found in this region. It is suggested that the granules represent the product of the Balbiani ring, possibly a messenger RNA bound to protein, and that they regularly pass into the cytoplasm through a narrow central channel in the pores of the nuclear envelope.     

Development of a binding assay for p53/HDM2 by using homogeneous time-resolved fluorescence

Using in situ hybridization techniques, a fixation step must precede denaturation to prevent disintegration of the chromosomes. The analysis of nuclei fixed by paraformaldehyde, preserving the native structure (three-dimensional or 3D fixation and analysis) has become possible with the development of confocal microscopy; however, the analysis of those fixed by methanol and acetic acid, dehydrating the nuclei (two-dimensional or 2D fixation and analysis), remains a very valuable tool for practical use in diagnostics and also in many cases for research. We compared both types of fixation and analyses using different cell lines and different DNA probes. Fixation of cells by methanol and acetic acid leads to the enlargement of contact of nuclei with the slide surface, resulting in a substantial increase of nuclear diameter, flattening of the nucleus, and consequently to a distortion of gene topology. Our results indicate that chromatin structures located in the outer parts of the nuclear volume (e.g., heterochromatin of some centromeres) are relatively shifted to the membrane of these nuclei, keeping the absolute centromere-membrane distance constant. On the other hand, euchromatin located in the inner parts of the nuclear volume is not shifted outside proportionally to the increase of molecular dimensions; consequently, the relative distances for the center of nucleus to gene are smaller after methanol-acetic acid fixation. The limitations of the analysis of dehydrated preparations for practical use and in research are discussed.     

Nuclear fragmentation and DNA degradation during programmed cell death in petals of morning glory (Ipomoea nil)

We studied DNA degradation and nuclear fragmentation during programmed cell death (PCD) in petals of Ipomoea nil (L.) Roth flowers. The DNA degradation, as observed on agarose gels, showed a large increase. Using DAPI, which stains DNA, and flow cytometry for DAPI fluorescence, we found that the number of DNA masses per petal at least doubled. This indicated chromatin fragmentation, either inside or outside the nucleus. Staining with the cationic lipophilic fluoroprobe DiOC₆ indicated that each DNA mass had an external membrane. Fluorescence microscopy of the nuclei and DNA masses revealed an initial decrease in diameter together with chromatin condensation. The diameters of these condensed nuclei were about 70% of original. Two populations of nuclear diameter, one with an average diameter about half of the other, were observed at initial stages of nuclear fragmentation. The diameter of the DNA masses then gradually decreased further. The smallest observed DNA masses had a diameter less than 10% of that of the original nucleus. Cycloheximide treatment arrested the cytometrically determined changes in DNA fluorescence, indicating protein synthesis requirement. Ethylene inhibitors (AVG and 1-MCP) had no effect on the cytometrically determined DNA changes, suggesting that these processes are not controlled by endogenous ethylene.     

Neutron diffraction of chromatin in interphase nuclei and metaphase chromosomes

We have used neutron diffraction to study chromatin structure in interphase nuclei and metaphase chromosomes as a function of decreasing ion concentration. Aliquots of a suspension of rat liver nuclei prepared in a polyamine-free buffer were washed in buffers of 1/3, 1/6 and 1/12 if the original concentration of monovalent and divalent cations (40 mM KCl; 20 mM NaCl; 1.2 mM MgCl2). After the first dilution step (1/1 to 1/3), only small changes occurred in the diffraction pattern. They can be interpreted by a loosening of the original structure, i.e. by the formation of isolated buffer-filled spaces with an overall size of the order of 35-45 nm. Drastic changes in the diffraction pattern were observed, however, when the nuclei were washed in the more diluted buffers (1/6 and 1/12). The profiles of the distances distribution functions indicate the formation of supranucleosomal particles with an overall diameter of 40-50 nm. The compact chromatin structure disassembled directly into these fundamental structural units. Structural transformations in the Chinese hamster ovary metaphase chromosomes were induced by diminishing the Ca2+ ion concentration of the buffer from originally 3.0 mM to 0.3 mM and/or by increasing the pH value of the buffer from originally 7.0 up to 8.0. The neutron diffraction patterns remained essentially unchanged during these treatments, i.e. the decondensation of the chromosomes as observed in the light microscope is not accompanied by disassembly at the ultrastructural level between 2 nm and 150 nm.     

Suggestive Structural Evidence for Macronuclear "Subnuclei" in Tetrahymena pyriformis GL

SYNOPSIS Structural changes in the Feulgen-positive material of the Tetrahymena pyriformis GL macronucleus have been observed during the cell cycle. From the finely granulated appearance in the interphase cell it appears as small rods, often arranged in pairs (probably the endomitotic stage) during early morphogenesis and as larger (and fewer) aggregates of granules during the nuclear division. These latter aggregates are also visible in dividing nuclei in the electron microscope where groups of chromation granules are separated by fairly empty nucleoplasm. It is suggested that these Feulgen positive aggregates in dividing nuclei are macronuciear segregation units or "subnuclei." The number per dividing macronucleus may vary from one experiment to another, but the variation seems to be related to cell volume. The distribution of the aggregates among the daughter nuclei is almost equal. The total number per dividing macronucleus is about 80 which is close to the estimated number of "subnuclei" in the T. pyriformis macronucleus (Allen and Nanney, 1958).
Some calculations are made on the polyploidy of the T. pyriformis GL macronucleus. Using published electron micrographs of micronuclei of known age to calculate the total number of chromatin granules per haploid nucleus, the polyploidy of the strain GL macronucleus is about 40. This figure is half of that expected from Allen and Nanney's estimation, since they assumed that the "subnuclei" were diploid; however, it is in agreement with the reported haploid nature of the "subnuclei" as found by Woodard, Gorovsky & Kaneshiro, 1968. Further calculations suggest that each macronuclear "chromosome" is composed of about 40 chromatin granules; an indication of such a chain arrangement of the chromatin granules has been observed in the phase contrast and electron microscope during the earliest macronuclear events, i.e., at the macronuclear "prophase."     

The role of H2B histones from the sea urchin sperm in the formation of supranucleosome structures

The structural role of histone H2B from sea urchin sperm (H2Bsp) has been examined in experiments on reconstitution of chromatin from DNA and core histones taken in three variants: (1) four core histones from sea urchin sperm; (2) four core histones from calf thymus; (3) (H3, H4, H2A) from calf thymus and H2Bsp. It is shown that H2Bsp when present in reconstituted chromatin induces its aggregation. Fidelity of the reconstitution of nucleosomes has been tested using DNase I probe, one- and two-dimensional electrophoresis and electron microscopy. The reconstitutes that contain H2Bsp appear under electron microscope mainly as regular closely spaced large granules, about 450 A in diameter, which are very similar to the granules found in "native" sea urchin sperm chromatin. The reconstitutes formed by four core histones from calf thymus appear as randomly arranged particles, about 100 A in diameter. We conclude that histone H2Bsp participates in interactions between nucleosomes and is involved in the formation of the condensed supranucleosomal structure in sea urchin sperm chromatin.     

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.     

Isolation of rosette-like structures from partially deproteinized chromatin in rat hepatocytes

The structure of partial deproteinized rat hepatocyte chromatin has been studied. Depending on the magnesium concentration the chromatin of isolated nuclei is present in the two conditions: diffuse (at 0-1.5 mM MgCl2) and condensed (at 2-5 mM MgCl2). The main components of nuclei with condensed chromatin are chromomers--globular structures about 100 nm in diameter. By treating such nuclei with heparin and dextransulfate one can observe a rosette-like structure with lateral loops having the following parameters: the length of the loops, 15-20 micron; the number of loops, 15-30. The rosette-like structures are sensitive to endogenous nuclease and DNase 1, but not to RNase. Pronase or higher concentration of polyanions give rise to unfolding of the rosette-like structures. The rosette structures cannot be isolated from the nuclei with diffuse chromatin. On the basis of these observations a hypothesis of chromatin structural organization in the interphase nucleus is proposed, and the connection of the rosette-like structures with some structural levels of chromatin organization is discussed.     

Studies on the role of histones H1 (f1) and H5 (f2c) in chromatin structure

Chicken erythrocyte and liver nuclei, isolated and fixed in isotonic saline, contained compact chromatin fibers about 200 Å in diameter. Fibers very similar in dimension and appearance are usually visible in thin sections of fixed nuclei in situ and probably represent chromatin organization close to the native state. After suspension of isolated nuclei in a mildly hypotonic buffer, chromatin fibers extended, became reduced in diameter and apparently unraveled in places. Under such conditions, new detail was revealed suggestive of both helical structure and of subunit organization. The fibers extended and contracted reversibly, changing in diameter from 200 Å to about 100 Å when alternately exposed to isotonic saline and to distilled water. The 200 Å fibers were irreversibly lost, however, following extraction of nuclei with high concentrations of NaCl which selectively removed H1 from liver and H1 plus H5 from erythrocyte chromatin. After extraction of more tightly complexed histones, the residual chromatin consisted mainly of fine filaments less than 30 Å thick. These results suggest a model for native chromatin fibers in which sub-unit organization and coiled configuration are combined, and in which histones H1 and H5 play an integral role in the maintenance of structure.