Structural properties of barley nucleosomes

The structural properties of barley oligonucleosomes are investigated and compared to those of rat liver oligomers. Extraction of barley chromatin was performed using mild nuclease digestion of isolated nuclei leading to a low ionic strength soluble fraction. Oligonucleosomes were fractionated on sucrose gradients and characterized for DNA and histone content. Physico-chemical studies (sedimentation, circular dichroism and electric birefringence) showed that barley oligonucleosomes exhibit properties very close to those of the H1-depleted rat liver counterparts. Moreover, in situ, barley linker DNA was more sensitive to micrococcal nuclease digestion than that of rat liver. These results suggest that barley oligonucleosomes show a less compact structure than their rat liver counterparts and appear to be in contradiction with the very condensed organization of barley chromatin previously suggested.     

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.     

Chemically induced carcinogenesis affecting chromatin structure in rat hepatocarcinoma cells

A new, chemically induced animal tumor cell line (HeDe) was established and characterized by its property of causing aggressively growing tumors in specific strain of rats and changes in the chromatin structure. Results show that (1) the nuclear material in nuclei of normal resting (G0) hepatocytes consists mainly of decondensed veil-like chromatin, chromosomes being clustered in six lobular domains; (2) nuclei of HeDe cells contain primarily supercoiled chromatin; or (3) the nuclear material of tumor cells undergoes apoptosis seen as apoptotic bodies. Heterogeneity of chromatin structures was expressed as contour/area ratio and was nine times higher in apoptotic cells and two times higher in tumor cells compared to resting cells.     

Chromatin maturation depends on continued DNA-replication

The structure of [3H]thymidine pulse-labeled chromatin in lymphocytes differs from that of non-replicating chromatin by several operational criteria which are related to the higher nuclease sensitivity of replicating chromatin. These structural features of replicating chromatin rapidly disappear when the [3H]thymidine pulse is followed by a chase in the presence of an excess of non-radioactive thymidine. However, when the rate of DNA replication is reduced, as in cycloheximide-treated lymphocytes, chromatin maturation is retarded. No chromatin maturation is observed when nuclei from pulse-labeled lymphocytes are incubated in vitro in the absence of DNA precursors. In contrast, when these nuclei are incubated under conditions known to be optimal for DNA replication, the structure of replicating chromatin is efficiently converted to that of 'mature', non-replicating chromatin. We conclude that the properties of nascent DNA and/or the distance from the replication fork are important factors in chromatin maturation.     

干旱胁迫对不同耐旱性大麦品种叶片超微结构的影响

选用耐旱性不同的3个大麦(Hordeum sativum)品种作为研究对象, 分析干旱胁迫对其叶肉细胞叶绿体、线粒体和细胞核超微结构的影响。结果表明, 3个大麦品种在非胁迫条件下其超微结构无明显差异。遭受干旱胁迫后, 不耐旱大麦品种Moroc9-75叶片细胞核中染色质的凝聚程度高, 叶绿体变形, 外被膜出现较大程度的波浪状和膨胀, 同时基粒出现弯曲、膨胀、排列混乱的现象; 线粒体外形及膜受到破坏、内部嵴部分消失等。耐旱大麦品种HS41-1叶片细胞中染色质虽出现凝聚, 但凝聚程度低; 其叶绿体及线粒体与非胁迫条件下基本相似, 多数未见明显损伤。耐旱中等的大麦品种Martin叶片超微结构的变化则介于二者之间。因此, 干旱胁迫下叶绿体外形、基粒和基质类囊体膜结构的完整性与基粒的排列次序、染色质的凝聚度和线粒体膜及嵴的完整性与大麦的耐旱性相关, 这些特性可作为评价大麦耐旱性强弱的形态结构指标。     

Higher order structure in a short repeat length chromatin

Polynucleosomes from calf brain cortical neurone nuclei have an average repeat length of less than 168 base pairs. The ability of this material to adopt higher order structure has been assessed by various physical techniques. Although containing on average less DNA per nucleosome than is required to form a chromatosome, this short repeat length chromatin folded in an H1 dependent manner to a structure with properties similar to those observed for longer repeat length chromatins such as that of chicken erythrocyte (McGhee, J.D., D.C. Rau, E. Charney, and G. Felsenfeld, 1980, Cell, 22:87-96). These observations are discussed in the context of H1 location in the higher order chromatin fiber.     

Properties of condensed chromatin in barley nuclei

A method for isolation and purification of intact nuclei from barley leaves was developed and several properties of the chromatin were studied. The dense structure of the main part of the chromatin does not alter the accessibility of the DNA to nucleases. 60% of the nuclear DNA can be degraded by micrococcal endonuclease. Nevertheless the solubility of the chromatin fragments depends on the extent of nuclease digestion; solubilisation occurring only when the major part of the internucleosomal DNA was degraded (30% of digestion). Electron microscopic observations suggest that this was due to particularly dense organization of the chromatin in situ. The possible physiological meaning of some of these properties are discussed.     

Partial characterization of nuclear binding sites for retinol delivered by cellular retinol binding protein

Retinol (vitamin A alcohol), which plays an important role in the differentiation of epithelia, can be transferred to chromatin in vitro. Rat liver chromatin can accept retinol in a specific and saturable manner only when the retinol is presented as a complex with cellular retinol-binding protein (CRBP). A partial characterization of the nuclear components responsible for accepting retinol is reported here. A preparation of solubilized chromatin isolated from liver nuclei was able to accept retinol from its complex with CRBP as described previously for nuclei and chromatin. The binding of retinol to chromatin was noncovalent. However, chromatin prepared from nuclei which were incubated with DNase I or micrococcal nuclease did not accept retinol specifically. Chromatin in the form of mono and dinucleosomes also did not accept retinol. However, treatment of nuclei with RNase did not affect the specific binding of retinol. Furthermore, it has been found that retinol was not transferred to purified double or single stranded DNA. These results are interpreted to indicate that the transfer of retinol to specific nuclear binding sites requires a higher order of chromatin structure than that occurring in nucleosome preparations.     

Iron overload and gene expression in HepG2 cells: analysis by differential display

A cell-free system derived from carrot cell cytosol extract has been developed for reassembling nuclear structure around the added demembranated sperm chromatin of Xenopus. Morphological evidence suggests that reassembled nuclei display the typical characteristics of normal eukaryotic nuclei, such as double-layered nuclear membrane and nuclear pores. Micrococcal nuclease treatment indicates that remodeling of the demembranated sperm chromatin has occurred and the structure of nucleosome is formed during nuclear reconstitution. These data indicate that the nuclear reconstitution can be induced in cell-free systems from plants, and the self-assembly of the nucleus is ubiquitous in both animal and plant cells.     

干旱胁迫对不同耐旱性大麦品种叶片超微结构的影响

选用耐旱性不同的3个大麦（Hordeum sativum）品种作为研究对象,分析干旱胁迫对其叶肉细胞叶绿体、线粒体和细胞核超微结构的影响。结果表明,3个大麦品种在非胁迫条件下其超微结构无明显差异。遭受干旱胁迫后,不耐旱大麦品种Moroc9-75叶片细胞核中染色质的凝聚程度高,叶绿体变形,外被膜出现较大程度的波浪状和膨胀,同时基粒出现弯曲、膨胀、排列混乱的现象;线粒体外形及膜受到破坏、内部嵴部分消失等。耐旱大麦品种HS41-1叶片细胞中染色质虽出现凝聚,但凝聚程度低;其叶绿体及线粒体与非胁迫条件下基本相似,多数未见明显损伤。耐旱中等的大麦品种Martin叶片超微结构的变化则介于二者之间。因此,干旱胁迫下叶绿体外形、基粒和基质类囊体膜结构的完整性与基粒的排列次序、染色质的凝聚度和线粒体膜及嵴的完整性与大麦的耐旱性相关,这些特性可作为评价大麦耐旱性强弱的形态结构指标。     

Cations and the accessibility of chromatin to nucleases.

When rat liver nuclei prepared with polyamines as stabilising cations are digested with DNAase II, release of both inactive chromatin and Mg-soluble, active chromatin is greatly reduced, in comparison to digestion of liver nuclei prepared with Mg2+ as stabilising cation. Chromatin release from polyamine stabilised nuclei is also inhibited relative to Mg-stabilised nuclei following digestion with micrococcal nuclease under two very different cation conditions. Nuclei prepared with polyamines and monovalent ions as stabilising cations exhibit properties intermediate between these two extremes with both nucleases. These effects are due to residual binding of polyamines to chromatin, which is thus maintained in a condensed state, inaccessible to nucleases. Since polyamine binding is not easily reversed, concentrations of polyamines and other cations must be rigidly controlled in experiments on chromatin structure if artefacts are to be avoided. The significance of these findings to the nature and properties of active chromatin within the intact nucleus is considered.     

Chromosome behaviour during meiotic prophase in the solanaceae

The molecular structure of chromatin during dogfish spermiogenesis was examined by electron microscopy after the dispersion of nuclei at low ionic strength. In early and late stages of differentiation (round and elongating spermatids), chromatin is globular, although basic nuclear proteins are different from those present in somatic nuclei. Three protein fractions are complexed with DNA in sperm nuclei. These fractions appear at the end of differentiation (elongated spermatids), subsequently undergoing a modification of their solubilization properties; only one protein fraction remains acid-soluble. Dispersed chromatin from sperm nuclei again shows a beads-on-a-string configuration both in the presence of the three specific sperm proteins and when the acid soluble fraction is extracted. Variations of the mean diameter of chromatin subunits during spermiogenesis appear rather limited compared to extensive modifications of chromatin superstructures.     

The study of chromatin and chromosome structure on preparations of interphase nucleus derivatives resulting from nuclear wall removal. IV. Structural heterogeneity of stretched chromatin in membrane-free cell nuclei from human peripheral lymphocytes

Densely aggregated chromatin of mature human or animal peripheral lymphocytes is inaccessible for structural investigation on preparations of both intact cell and conventionally spread chromatin. Giemsa- and DAPI-positive "free chromatin" structures, in addition to amembraneous nuclei, were isolated from intact lymphocytes gently treated with Triton-X-100. Surface stretching of both these nuclei and structures, shortly fixed in methanol-glacial acetic acid (3:1), revealed three main types of these "free chromatin" structures: dense chromatin structures (DCS), loose chromatin structures (LCS) and nuclear spreads (NS). The share of each nuclear derivative may be shifted by changing either detergent concentration and(or) the time of incubation in detergent solution. Each DSC consists of condensed "residual" nucleus, similar in from and size with an intact lymphocyte nucleus, and involves 1-15 uni- or olygonemic chromatin sprouts of different length. LSC contain heterogeneously loosened spindle-shape or drop-like nuclei, being several times longer and wider than DCS-nuclei, and 1-3 long uni- or olygonemic chromatin tail-pieces and incidentally observed lateral chromatin sprouts. The majority of LCS contain either a chromocenter of different number of end-to-end associated spindle-shape domains of condensed chromatin. The latter reached 2-5 x 1.5 microns being cross-striated or spiral in structure. NS represent spread chromatin fibrillar structures varying from 150 to 500 microns in length and from 1.5 to almost 50 microns in width. NS consist of 0.3-0.4 micron smooth and 0.4-0.8 micron beaded chromatin fibres. Thin fibres produce web-like domains of NS. and thick fibres form olygonemic bundles or end-to-end association of unit chromatin fibres within NS. Some portion of thick unit fibres of NS gave rise to local splitting into two thin fibres with a similar bead patterns. Thick argyrophilic fibers of the nucleolus also displayed a beaded structure and commonly spread hand-in-hand with the basic chromatin fibre aggregations.     

Study of the effect of different concentrations of a detergent on the chromatin structure of Physarum polycephalum during the mitotic cycle

The influence of different concentrations of detergent Joy on the chromatin structure of Physarum polycephalum in the process of mitotic cycle was studied electronmicroscopically. The investigations showed that at Joy concentrations less than 0,01% a small part of nuclei disrupt and, as a rule, chromatin is insufficiently dispersed; at concentrations more than 0,1% the detergent may influence the chromatin structure of Physarum polycephalum. Based on the data obtained we consider that the optimal detergent concentrations that practically do not influence the chromatin structure and lead to disruption of the majority of nuclei and to the proper dispersion of chromatin is 0,1-0,01%.     

An in vitro reconstitution system for the assessment of chromatin protein fluidity during Xenopus development

Chromatin fluidity, which is one of the indicators of higher-order structures in chromatin, is associated with cell differentiation. However, little is known about the relationships between chromatin fluidity and cell differentiation status in embryonic development. We established an in vitro reconstitution system that uses isolated nuclei and cytoplasmic extracts of Xenopus embryos and a fluorescence recovery after photobleaching assay to measure the fluidities of heterochromatin protein 1 (HP1) and histone H1 during development. The HP1 and H1 fluidities of nuclei isolated from the tailbuds of early tadpole stage (stage 32) embryos in the cytoplasmic extracts of eggs and of late blastula stage (stage 9) embryos were higher than those in the cytoplasmic extracts of mid-neurula stage (stage 15) embryos. The HP1 fluidities of nuclei isolated from animal cap cells of early gastrula stage (stage 10) embryos and from the neural plates of neural stage (stage 20) embryos were higher than those isolated from the tailbuds of stage 32 embryos in egg extracts, whereas the HP1 fluidities of these nuclei were the same in the cytoplasmic extracts of stage 15 embryos. These results suggest that chromatin fluidity is dependent upon both cytoplasmic and nuclear factors and decreases during development.     

Superstructural differences between chromatin in nuclei and in solution are revealed by kinetics of micrococcal nuclease digestion.

Digestion of chromatin in nuclei by micrococcal nuclease, measured as the change in the concentration of monomer-length DNA with time, displays Michaelis-Menten kinetics. Redigestion of soluble chromatin prepared from nuclei by micrococcal nuclease treatment, however, is apparently first order in enzyme and independent of chromatin concentration. This qualitative difference results from an increase in the apparent second order rate constant, kcat/Km, for liberation of monomer DNA: the apparent Km for soluble chromatin is lower by close to 3 orders of magnitude than that for chromatin in nuclei, whereas kcat decreases by less than 1 order of magnitude. Neither the integrity of the nuclear membrane nor the presence of histone H1 contributes to the high Michaelis constant characteristic of chromatin in nuclei. Moreover, differences due to the buffers used for digestion and redigestion are minimal. Low catalytic efficiency is, however, correlated with the presence of higher order chromatin superstructure. Micrococcal nuclease added to soluble chromatin under nondigesting conditions at low ionic strength (I = 0.002) co-sediments with chromatin in sucrose gradients. In 0.15 M NaCl, added nuclease no longer sediments with chromatin and redigestion kinetics become first order in both enzyme and substrate. Kinetic analysis of this type may afford an assay for native, higher order structures in chromatin. Our results suggest that micrococcal nuclease binds to soluble chromatin through additional interactions not present in nuclei, which may be partly ionic in nature.     

Cytophotometric analysis of the chromatin structural conformity in interphase nuclei detected in UV light and by gallocyanine staining

Geometric and optical parameters of chromatin of hepatocyte nuclei have been examined before (UV, lambda = 265 nm) and after gallocyanine staining. Quantitative parameters of the chromatin structure in the same nuclei measured in situ by a scanning microscope-photometer (step size 0.125 micron) before and after staining were equal. Tinctorial properties of chromatin granules (condensed part of the nuclear material) and its diffuse part were different. It is suggested that the difference between granules and the nongranular part of chromatin is not only of optical but also of chemical nature.     

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.