AN ANALYSIS OF HETEROCHROMATIN IN MAIZE ROOT TIPS

The B chromosomes of maize are condensed in appearance during interphase and are relatively inert genetically; therefore they fulfill the definition of heterochromatin. This heterochromatin was studied in root meristem cells by radioautography following administration of tritiated thymidine and cytidine, and was found to behave in a characteristic way, i.e. it showed asynchronous DNA synthesis and very low, if any, RNA synthesis. A cytochemical comparison of normal maize nuclei with nuclei from isogenic maize stock containing approximately 15–20 B-chromosomes in addition to the normal complement has revealed the following: (a) the DNA and histone contents are greater in nuclei with B chromosomes; (b) the proportion of DNA to histone is identical with that of nuclei containing only normal chromosomes; (c) the amount of nonhistone protein in proportion to DNA in interphase is less in nuclei with B chromosomes than in normal nuclei. In condensed B chromosomes the ratio of nonhistone protein to DNA is similar to that in other condensed chromatin, such as metaphase chromosomes and degenerating nuclei. The B chromosomes appear to have no effect on nucleolar RNA and protein. Replication of B chromosomes is precisely controlled and is comparable to that of the ordinary chromosomes not only in synthesis for mitosis but also in formation of polyploid nuclei of root cap and protoxylem cells.     

FINE STRUCTURAL ALTERATIONS OF INTERPHASE NUCLEI OF LYMPHOCYTES STIMULATED TO GROWTH ACTIVITY IN VITRO

This report describes fine structural changes of interphase nuclei of human peripheral blood lymphocytes stimulated to growth by short-term culture with phytohemagglutinin. Chromatin is found highly labile, its changes accompanying the sequential increases of RNA and DNA synthesis which are known to occur in lymphocyte cultures. In "resting" lymphocytes, abundant condensed chromatin appears as a network of large and small aggregates. Early in the response to phytohemagglutinin, small aggregates disappear during increase of diffuse chromatin regions. Small aggregates soon reappear, probably resulting from disaggregation of large masses of condensed chromatin. Loosened and highly dispersed forms then appear prior to the formation of prophase chromosomes. The loosened state is found by radioautography to be most active in DNA synthesis. Small nucleoli of resting lymphocytes have concentric agranular, fibrillar, and granular zones with small amounts of intranucleolar chromatin. Enlarging interphase nucleoli change chiefly (1) by increase in amount of intranucleolar chromatin and alteration of its state of aggregation and (2) by increase in granular components in close association with fibrillar components.     

THE FINE STRUCTURE OF THE NUCLEI OF TETRAHYMENA PYRIFORMIS THROUGHOUT THE CELL CYCLE

The fine structure of the nuclei of logarithmically growing Tetrahymena pyriformis, strain HSM, was studied at 30-minute intervals throughout the cell cycle. Organisms were selected at similar stages of cytokinesis by means of a braking pipette, incubated, fixed in OsO₄, and embedded in agar to facilitate subsequent preparation for electron microscopy. Aggregates of micronuclear chromatin underwent a decrease in density and number with a concomitant increase in size throughout interphase. There were no impressive changes in macronuclear morphology. It was found possible to estimate a cell's progress through interphase by observation of micronuclear morphology, but attempts to correlate changes in fine structure with periods of DNA synthesis were unsuccessful.     

The ultrastructure and properties of centromeric heterochromatin in cultured bovine trachea cells]

The fine organization of the centromere structural heterochromatin (CSH) in a cell culture of calf trachea (TR) was studied by the methods of light and electron microscopy after fixation in native conditions and after treatments with water Henk's solutions and solutions of Ca2+ of different concentrations. In interphase nuclei the CSH forms compact blocks--the chromocentres, which are connected with the nuclear envelope or the nucleolus. The diameter of the main class of DNP fibers in the CSH, chromosomal arms and chromocentres after fixation of control cells and after treatment with 50% Henk's solution is 20-25 nm. 10-15 nm DNP fibers are largely found in the contacts with kinetochores. After 20% Henk's solution treatment 10-15 nm fibers predominate in the CSH and chromosomal arms. A chromonema--a 100 nm chromatin fiber--is detected in the CSH after treatments with different concentrations of Ca2+ solutions, as well after fixation in native conditions. The peculiarity of structural organization and properties of the calf CSH at interphase and mitosis as compared with analogous regions in mouse chromosomes are suggested to be connected with the composition of its DNA and proteins.     

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.     

The similarity of DNA sequences remaining bound to scaffold upon nuclease treatment of interphase nuclei and metaphase chromosomes.

The fragments of DNA attached to protein skeleton of interphase nuclei or metaphase chromosomes were obtained. Both the method involving restriction endonuclease treatment/1,2/and a novel procedure based on mild staphylococcal nuclease digestion were used. In the latter case, DNA fragments remaining bound to nuclei or chromosomes are not enriched in satellite but only in abundant middle repetitive DNA. The shorter the fragments of attached DNA, the higher the content of middle repetitive DNA in the fraction. It has a slightly higher density in a CsCl gradient comparing to the main DNA. The yield of attached DNA, its distribution in a CsCl density gradient, and its renaturation properties are essentially the same for interphase and metaphase chromosomes. The average size of DNA loops was found to be equal to approximately 60 kb for both metaphase chromosomes and interphase nuclei. The conclusion has been drawn that the bulk of attachment sites of DNP fibrils to axial chromosomal structures remains unchanged during the cell cycle.     

The state of the chromosomes in the interphase nucleus

In the living interphase nucleus no chromosomal structures are visible. Yet in the injured cell and after treatment with most histological fixatives chromatin structures become apparent. Under certain conditions this appearance of structure in the living interphase nucleus is reversible. We have found that this change in the interphase nucleus is the result of a change in the state of the chromosomes. In the living nucleus the chromosomes are in a greatly extended state, filling the entire nucleus. Upon injury the chromosomes condense and therefore become visible. At the same time the nuclear volume decreases. This behavior of the chromosomes is connected with their content of desoxyribonucleic acid (DNA). This view is based on the following observations: (a) Distribution of DNA in the Nucleus.-(1) The living interphase nucleus of uninjured cells absorbs diffusely at 2537 A. No chromosomal structures are visible in ultraviolet photographs unless they are also distinct in ordinary light. If the chromosomes are made to condense they become visible and the absorption at 2537 A is now localized in these structures. (2) After fixation with formalin and osmic acid interphase nuclei stain diffusely with Feulgen. These fixatives preserve the extended state of the chromosomes. (3) If nuclei are teased out in non-electrolytes (sucrose, glycerin) the chromosomes are extended. Such nuclei stain homogeneously with methyl green. On adding salts the chromosomes condense and the methyl green is now restricted to the visible structures. (b) Extension and Condensation of Isolated Chromosomes.-When chromosomes isolated from interphase nuclei of calf thymus are suspended in sucrose, their volume is four to five times larger than in saline, but they retain their characteristic shapes. Chromosomes from which DNA and histone have been removed do not show this reversible extension and condensation, neither do lampbrush chromosomes of frog oocytes which contain very little DNA. During mitosis a partial condensation of the DNA occurs in prophase, so that the mitotic chromosomes now occupy a much smaller volume of the nucleus. At telophase the chromosomes swell again to fill the entire nucleus.     

Do individual allocyclic chromosomes in metaphase reflect their interphase domains?

Summary Individual S phase allocyclic chromosomes have been analyzed in Bloom syndrome lymphocytes, in cells with an r(9), and in hypotetraploid Ehrlich mouse ascites cells treated with 1-methyl-2-benzyl hydrazine. On the basis of the following observations, we conclude that such chromosomes more or less reflect their domains in interphase: (1) The S phase allocyclic chromosomes have the same structure as S phase prematurely condensed chromatin (PCC) in fused cells; in other words they form limited areas of chromatin dots; (2) the allocyclic chromosome is the only chromosome in a metaphase plate which synthesizes DNA simultanneously with interphase nuclei; (3) the size of the allocyclic chromosomes is related to the size of the corresponding metaphase chromosome; and (4) the S phase allocyclic chromosomes resemble closely the chromosome domains in interphase made visible with biotinylated human DNA. A variety of evidence shows that most allocyclic chromosomes are simply left behind in their cycle, which presumably is caused by a deletion or inactivation of a hypothetical coiling center situated on each chromosome arm.     

大麦细胞核及染色体肌球蛋白免疫细胞化学定位

对去除DNA、组蛋白和大部分非组蛋白的大麦(Hordeum vulgare)细胞核和染色体间接免疫荧光标记实验结果表明:抗肌球蛋白抗体的荧光标记弥散分布在整个细胞核和染色体上;进一步应用免疫胶体金技术分析肌球蛋白在细胞核和染色体的分布情况,发现在染色体中散布着大量的胶体金颗粒;间期细胞核中胶体金颗粒主要分布在核仁和染色质中。上述实验结果表明:肌球蛋白是细胞核及染色体非组蛋白组成成分。本文还对肌球蛋白在细胞核和染色体中的分布规律进行了讨论。     

小鼠生精细胞染色质与染色体构筑的扫描电镜研究(简报)

正常情况下,染色质和染色体在细胞内呈高度致密状态,在光镜和透射电镜下常呈浓染的斑块状。由于方法学上的困难,至今对染色质乃至染色体的微细结构,仍缺乏清楚的了解。特别是关于染色质如何凝缩形成染色体方面,现仍存在有争论。扫描电镜的冷冻割断技术,曾被用于对游离细胞间期核染色质的观察,并取得了较好的     

Interphase chromosomal deoxyribonucleoprotein isolated as a discrete structure from cultured cells

A new procedure is described for the preparation of interphase chromatin from cultured mouse cells (line P815). The primary objective of this procedure was to eliminate exchanges of histones between deoxynucleoprotein molecules; this objective is shown experimentally to have been attained. The chromatin is released from cells by the non-ionic detergent Nonidet P40 in medium of low ionic strength (0.1 mM-KNa₂PO₄), and may then be sedimented as a structure which conserves the general form and ultrastructural characteristics of chromatin within the cell. The nuclear envelope cannot be detected in these structures by electron microscopy, and their content of choline-containing phospholipids is less than 10% of that of nuclei. The maintenance of form in this structure must thus depend on properties of the chromatin itself, and possibly on the more compact peripheral chromatin.Soluble DNP² prepared by shearing these structures has the same relative contents of DNA, histones, non-histone proteins and RNA as DNP prepared by standard methods. Analyses by electrophoresis on polyacrylamide gels of the non-histone proteins reveals certain differences from the pattern of these proteins in DNP prepared by a salt precipitation method. The template activity for RNA synthesis, in the presence of Escherichia coli RNA polymerase of sheared, soluble DNP prepared by this procedure, is comparable to that of DNP prepared by other methods. However, in the absence of exogenous RNA polymerase the rate of RNA synthesis by structured (unsheared) chromatin is about ten times higher than the rate using sheared DNP.The rapid removal of the nuclear envelope in this lysis procedure allowed experimental examination of the origin of the histones and non-histone proteins of DNP. When DNP was prepared from a mixture of two populations of cells, one containing DNA distinguishable by a density label and the other containing radioactively labelled proteins, radioactive proteins were found exclusively in DNP of normal density, and not in dense DNP and vice versa. It is concluded that the proteins of DNP prepared in this way are not acquired during the preparation procedure but were already associated with DNA in vivo, and that other proteins are not bound non-specifically to DNA during the preparation of DNP. When a mixture of DNP molecules prepared, in this way is precipitated in 150 mm-NaCl and redissolved, some radioactively labelled histones migrate onto dense DNA molecules.This procedure is suitable for routine, quantitative isolation of chromosomal DNP from small numbers of cells; it is also applicable to cells of other cultured lines.     

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.     

Dynamic inorganic ion-protein interactions in structural organization of DNA of living cell nuclei.

Staining polarization optical techniques showed differences in the structural organization of DNA of chromatin in interphase nuclei and in mitotic chromosomes. The DNA was non-birefringent in intact interphase cell nuclei, but birefringent in chromosomes and in isolated nuclei incubated in a physiological electrolyte solution. The birefringence of DNA appears to be related to an unfolding of DNA filaments induced by free cations and to the oriented binding of dye molecules to DNA phosphates. We propose that the actual concentration of free cations inside the living cell nuclei is regulated by a dynamic interaction between nuclear proteins and ions.     

多头绒泡菌间期细胞核和中期染色体中的银染蛋白分析*

电镜原位观察结合图象分析研究了多头绒泡菌Physarum polycephalum Schw间期细胞核和中期染色体中银染蛋白的形状、大小和分布。结果看到,银染蛋白主要呈颗粒状存在于间期细胞核和中期染色体中。银粒的大小不一,分布不均匀。间期细胞核中存在众多直径在5~15nm的银粒,其中10nm以上的较大银粒主要分布于核仁,集缩染色质和核基质部分10nm以上银粒不多。中期细胞核内10nm以上的较大银粒主要分布于染色体中。染色体中除含有一些较大银粒外,多数银粒的直径为5~10nm。本文结果提示,构成染色体骨架的嗜银蛋白可能来自间期细胞核的染色质、核基质和核仁。     

Features of interactions of p68 anchorosomal protein with the nuclear envelope in the cell cycle

Chromatin associated with the nuclear envelope appears in the interphase nuclei as a layer of anchorosomes, granules 20-25 nm in diameter. The fraction of chromatin directly associated with the nuclear envelope is resistant to decondensation, shows a low level of DNA methylation, and contains specific acid-soluble proteins. However, mechanisms underlying the interaction of chromatin with the nuclear envelope are not fully understood. Specifically, it is not known whether anchorosomes are permanent structures or if they undergo reversible disassembly during mitosis, when contacts between chromatin and the nuclear envelope are destroyed. We obtained immune serum recognizing a 68 kDa protein from the nuclear envelopes fraction and studied the localization of this protein in interphase and mitotic cells. We show that this protein present in the NE/anchorosomal fraction does not remain bound with chromosomes during mitosis. It dissociates from chromosomes at the beginning of the prophase and then can be identified again at the periphery of the newly forming nuclei in the telophase.     

Evidence for the organization of chromatin in megabase pair-sized loops arranged along a random walk path in the human G0/G1 interphase nucleus

We determined the folding of chromosomes in interphase nuclei by measuring the distance between points on the same chromosome. Over 25,000 measurements were made in G0/G1 nuclei between DNA sequences separated by 0.15-190 megabase pairs (Mbp) on three human chromosomes. The DNA sequences were specifically labeled by fluorescence in situ hybridization. The relationship between mean-square interphase distance and genomic separation has two linear phases, with a transition at approximately 2 Mbp. This biphasic relationship indicates the existence of two organizational levels at scales > 100 kbp. On one level, chromatin appears to be arranged in large loops several Mbp in size. Within each loop, chromatin is randomly folded. On the second level, specific loop-attachment sites are arranged to form a supple, backbonelike structure, which also shows characteristic random walk behavior. This random walk/giant loop model is the simplest model that fully describes the observed large-scale spatial relationships. Additional evidence for large loops comes from measurements among probes in Xq28, where interphase distance increases and then locally decreases with increasing genomic separation.     

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.     

Different sensitivity of DNA in situ in interphase and metaphase chromatin to heat denaturation

Heat denaturation of DNA in situ, in unbroken cells, was studied in relation to the cell cycle. DNA in metaphase cells denatured at lower temperatures (8 degrees-10 degrees C lower) than DNA in interphase cells. Among interphase cells, small differences between G1, S, and G2 cells were observed at temperatures above 90 degrees C. The difference between metaphase and interphase cells increased after short pretreatment with formaldehyde, decreased when cells were heated in the presence of 1 mM MgCl2, and was abolished by cell pretreatment with 0.5 N HCl. The results suggest that acid-soluble constituents of chromatin confer local stability to DNA and that the degree of stabilization is lower in metaphase chromosomes than in interphase nuclei. These in situ results remain in contrast to the published data showing no difference in DNA denaturation in chromatin isolated from interphase and metaphase cells. It is likely that factors exist which influence the stability of DNA in situ are associated with the super-structural organization of chromatin in intact nuclei and which are lost during chromatin isolation and solubilization. Since DNA denaturation is assayed after cell cooling, there is also a possibility that the extent of denatured DNA may be influenced by some factors that control strand separation and DNA reassociation. The different stainability of interphase vs. metaphase cells, based on the difference in stability of DNA, offers a method for determining mitotic indices by flow cytofluorometry, and a possible new parameter for sorting cells in metaphase.