Configurational changes in rat liver nuclear chromatin and nucleoli caused by dissociation and reassociation of F1 histone.

Isolated rat liver nuclei treated in 5% perchloric acid (PCA) showed characteristic configurational changes causing a “lace-like” appearance of chromatin. The nucleoli lost their fine granular and fibroreticular structure, resulting in a “coarse granular” configuration. When extracts in 5% PCA were reassociated with the treated nuclei, the chromatin and nucleoli returned to their initial structure. Gel electrophoresis studies revealed that the extracts contained only F 1 histone, that F 1 histone was completely released from the nuclei, and that dissociated F 1 histone reassociated with F 1 histone-depleted nuclei. Chromatin isolated from F 1 histone-depleted nuclei lost the “knob-like” structures, which characterize native chromatin fibers, becoming smooth and fine threads. Chromatin fibers isolated from the reconstituted nuclei recovered their original “knob like” structures. This suggests that F 1 histone plays a crucial role in chromatin and nucleolus structural organization.     

Electron microscopic study of endopolyploid nuclei in rat trophoblast giant cells. IV. Changes in nucleolar ultrastructure during cell differentiation

The ultrastructure of the nucleolus of highly differentiated trophoblast giant cells has been studied on the 17th day of the foetus development. Changes in its morphology have been followed in relation to the degree of nuclear chromatin condensation and to the cell differentiation level. The nucleoli have a reticular structure in the nuclei with dispersed and condensed chromatin. In both the cases the nucleoli involve the four components: fibro-granular, fibrillar (of moderate and normal density) and lacunar regions; fibrillar centres are distinguished within the regions. In the nucleoli with condensed chromatin, unlike those with dispersed chromatin, the perinuclear chromatin is clearly seen, and the penetration of nucleolus-organizer threads along lacunae and deep into the nucleolus can be easily followed. The fibrillar centres are more obvious. With the run of a progressive differentiation of the trophoblast cells, the number of granules is reduced; first, the fibro-granular component covers a significant part of the nucleolus, then granules become visible only in the cortical zone of the nucleolus; in the nuclei with strongly condensed chromatin no granules are seen in the nucleolus.     

四膜虫(Tetrahymena shanghaiensis)大核核仁与去膜大核能诱导非细胞体系核重建

外源DNA或染色质在非洲爪蟾卵提取物中可以诱导细胞核样结构的重建。重建核除不具有核仁样结构外,在其它形态结构上与真核细胞核十分相似。前人的工作表明在重建核中具有核仁前体结构。但可能是由于缺少活性核仁组织者的缘故,这些核仁前体不能相互融合形成新生核仁。那么活性核仁组织者在重建核中是否能发挥其功能呢?为了研究这一问题,我们提取纯化了四膜虫的大核与大核的周边核仁。进一步去除大核的核被膜,并将去除核被膜的大核与大核核仁分别加入非洲爪赡卵非细胞体系中。通过电镜超薄切片观察,我们发现无论是与大核染色质相连的周边核仁还是分离纯化的核仁结构在非洲爪赡卵非细胞体系中都不能保持其原有结构特征,而是发生了典型核重建变化,并且在诱导形成的重建核中也看不到核仁样结构。这些结果说明具有活性的核仁组织者在加入非洲爪蟾卵提取物后既不能继续保持其原有的RNA转录功能也不能诱导新的核仁的出现。     

Cytological characteristics of the gonocytes in the course of their migration in early rat embryos]

The work was carried out in 23 rat embryos from 9,5 to the 11th day of development. In 9,5-day embryos the primary sex cells are localized in the mesenchyma of the allantois and in the intestinal entoderm. Later they migrate either with the blood flow or on the surface of cellular layers towards gonad germs which are reached by the 11th day of the intrauterine development. In the course of this process there occur structural and cytochemical changes in gonocyte nuclei. The nucleolus is replaced to the periphery of the nucleus, around it there appears a rim intensively stained with methylene green. Chromatin has a shape of thin threads. These changes of the nuclei seem to be associated with an increased synthesis of r-DNA and hence with the synthesis of substances having the role to prevent the somatic differentiation of primary sex cells in the course of migration.     

STUDIES ON ISOLATED NUCLEI : II. Isolation and Chemical Characterization of Nucleolar and Nucleoplasmic Subfractions

This paper describes the subfractionation of nuclei isolated from guinea pig liver by the procedure presented in the first article of the series (8). Centrifugation in a density gradient system of nuclear fractions disrupted by sonication permits the isolation of the following subfractions: (a) a nucleolar subfraction which consists mainly of nucleoli surrounded by a variable amount of nucleolus-associated chromatin and contaminated by chromatin blocks derived primarily from von Kupffer cell nuclei; (b) and (c), two nucleoplasmic subfractions (I and II) which consist mainly of chromatin threads in a coarser (I) or finer (II) degree of fragmentation. The protein, RNA, and DNA content of these subfractions was determined, and their RNA's characterized in terms of NaCl-solubility, nucleotide composition, and in vivo nucleotide turnover, using inorganic ₃₂P as a marker. The results indicate that there are at least three types of RNA in the nucleus (one in the nucleolus and two in the nucleoplasm or chromatin), which differ from one another in NaCl-solubility, nucleotide composition, turnover, and possibly sequence. Possible relations among these RNA's and those of the cytoplasm are discussed.     

Fragmentation of polyploid nuclei in the giant cells of the rat trophoblast. I. The ultrastructure of the nuclear fragments

Electron microscope study of the nuclear fragments in the rat trophoblast has demonstrated that the division of the trophoblast giant nucleus results first in the formation of a multinuclear cell. Each nuclear fragment is covered with its own nuclear envelope made of two membranes with numerous pore complexes. The chromatin in these nuclear fragments is condenced with various degrees of condensation, which depends on the step of placenta development, cell differentiation and the degree of nuclear fragmentation. The nuclear ultrastructure in nuclear fragments also depends on the degree of nuclear fragmentation and on the level of chromatin condensation. The nucleolus has no granular component. On large fragments, with lower chromatin condensation the nucleolus is not homogenous being made of fragments of more and of less electron dense fibrilles. Small light lacunae are seen in the nucleolus where chromatin threads and strands pass on. With a high chromatin condensation in the nucleus, round small nucleoli look homogenous being made of moderately electron dense fibrilles. Products of chromosome activity have been found in the nuclear fragments: accumulations of minute granules (d = 15--20 nm), perichromatinous granules (d = 35--40 nm), and fibrillar nucleolus-like bodies. In the multinuclear cell, made as the result of fragmentation of the initially giant nucleus, all the small nuclei are first arranged very close to each other, so that the contours of the neighbouring nuclei coincide.     

Uptake of ribosomal proteins by isolate HeLa nuclei.

The uptake of radioactive ribosomal proteins by isolated HeLa cell nuclei has been studied. Ribosomal proteins are taken up by nuclei $\text{in vitro}$ more rapidly than are cytosol proteins, suggesting that the uptake is selective. In addition, the ribosomal proteins are found associated with the nucleolus to a greater extent than are the cytosol proteins.     

Nuclear recovery from centrifugation-caused elongation: Involvement of the microfilament system in the nuclear plasticity

The plasticity of elongated nuclei with thread-like basal protrusions was investigated after centrifuging protonemal cells of the fernAdiantum capillus-veneris basipetally for 2 or 3 hr. The morphological recovery of the nuclei including the shortening process of the thread could directly be visualized by video microscopy of nuclei with bubble-like thread ends in centrifuged, living cells. The shortening proceeded in three phases: (1) the fast shortening of the part between the bubble and the nuclear apical main body (NAMB), (2) the slow shrinking of the bubble, (3) the entrance of the nucleolus into the NAMB. Although the thread shortening process was quite uniform, there were irregularities like reextension of the threads over short distances. The experimental system of elongated nuclei was used to probe the role of the cytoskeleton in the nuclear plasticity. Directly after basipetal centrifugation, thick strands of microfilaments (MFs) were found to be aligned with the nuclear threads, whereas microtubules (MTs) were not. In cytoskeleton-depolymerizing experiments, cytochalasin B caused a reduction of the shortening process, showing that the MF system in the cytoplasm is involved in the nuclear recovery. In non-centrifuged as well as in centrifuged cells, on the other hand, cytoplasmic streaming was efficiently inhibited by cytochalasin B, whereas it was not significantly affected by colchicine. The moderate effect of cytochalasin B on the thread shortening suggests that still other driving forces such as tension in the nuclear envelope and perhaps intranuclear forces are involved in the thread shortening mechanism.     

COMPOSITION OF RIBONUCLEIC ACID FROM VARIOUS PARTS OF SPIDER OOCYTES

Microphoretic purine-pyrimidine analyses of the ribonucleic acid (RNA) in nucleoli, nucleoplasm, cytoplasm, and yolk nuclei of spider oocytes have been carried out. The material necessary for the analyses was isolated by micromanipulation. Determinations of the amounts of RNA in the different parts of the cell were also performed. No differences between the composition of RNA in the nucleolus and the cytoplasm could be disclosed. Nucleoplasmic RNA was, on the other hand, distinctly different from that in the nucleolus and in the cytoplasm. The difference lies in the content of adenine, which is highest in nucleoplasmic RNA. The few analyses carried out on yolk nuclei showed their RNA to be variable in composition with a tendency to high purine values. The cytoplasm contains about 99 per cent of the total RNA in these cells, the nucleoplasm about 1 per cent, and the nucleolus not more than 0.3 per cent, although the highest concentrations are found in these latter structures. When considered in the light of other recent findings the results are compatible with the view that nucleolar RNA is the precursor of cytoplasmic RNA.     

The Fusion of Male and Female Nuclei in Fertilization of Higher Plants

Studies on the fusion of male and female nuclei in fertilization of Helianthus an- nuus L., Triticum aestivan L., Gossypium hisutum L., Hosta caerulea Tratt., and Pinus tabulaeformis Carr. were made in the present work. The results are summarized as follows: 1. The essential process of the fusion of male and female nuclei during syngamy in four species of angiosperms studied may' be generalized as follows: (1) the male nucleus made contact with the female one, (2) followed by the fusion of nuclear membranes between the male and female nuclei. (3) then the despiralization of male spireme happened and male nucleolus made its appearance inside of the fertilized egg nucleus (4) the male chromatin dispersed and make its appearance indistinguishable from that of the female chromatin, (5) the male and female nucleoli fused together to form a larger nucleolus as a sign of completion of the fusion of the two nuclei. In the first mitotic division of the zygote there was only one common mitotic spindle. 2. The essential process of the fusion of egg and sperm nuclei during syngamy in a gymnosperm-Pinus tabulaeformis could also be outlined as follows: (1) the sperm nucleus made contact with the egg nucleue, (2) the fusion of nuclear membranes happened between the male and female nuclei, (3) the male and female ehromatins condensed to form two separate groups of chromatin threads together with the very apparent apperance of the male and female nucleoli at this stage, (4) the male and female chromosomes grouped respectively in their own spindles while both nucleoli disappeared, (5) then the two spindles fused together and all the chromosomes arranged to form a common equatorial plate, (6) finally two daugter nuclei resulted from the mitotic division. 3. Based on the facts that there were two different patterns of the fusion of male and female nuclei in fertilization discribed, all of these accounts are in general accord with the condition usually described that there are two types of fertilization, the pre- mitotic and postmitotie syngamy in higher plants. The type of angiosperm fertilization and the mechanism of promoting the zygote to divide after fertilization are discussed, and the nuclear fusion in sexual reproduction has been compared with that of somatic cell hybridization.     

PROTEIN SYNTHESIS BY ISOLATED PEA NUCLEOLI

A new method is described for the preparation of active, nucleus-free nucleoli and chromatin in relatively high purity and in sufficient quantities to permit biochemical and electron microscopic investigation. This method consists of disintegrating previously isolated nuclei by grinding with glass beads in an isotonic medium thus liberating structurally intact nucleoli and chromatin threads. Nucleoli and chromatin are then purified by differential centrifugation in Ficoll solutions. A study of the chemical composition, submicroscopic structure, and biological activity of the nucleolar preparation has been made. An equivalent study of the chromatin material has also been carried out in order to assess the significance of chromosomal contamination in nucleolar protein synthesis. The isolated nucleoli rapidly incorporate leucine-C¹⁴ into acid and base stable compounds in vitro. Such incorporation lasts for 20 minutes at 37°C and is enhanced by the addition of an energy-regenerating system and a complete amino acid mixture. It is independent of the nuclear Ph 5 enzymes. The bulk of the incorporated label is recovered in the residual, ribosome-like nucleolar protein fraction and a small percentage is found in the acid-extractable basic proteins. The rate of protein synthesis by isolated nucleoli is more rapid than that occurring in the chromatin fraction. This is taken as an additional proof that the nucleolus is the principal site of protein synthesis in the interphase pea nucleus.     

The histological localization of DNA in rabbit medullary neurons

The nuclei of unfixed isolated rabbit neurons cleared on incubation with DNAse (10 mg/ml), but not RNAse (10 mg/ml). The nuclei stained for DNA with eight chromosomal or nuclear stains more intensely than the cytoplasm, and less intensely after treatment with DNAse (10 mg/ml). On the other hand, when the whole tissue was embedded and sectioned, DNA did not appear to be stained in the nucleus; the nucleolus and the cytoplasm were more heavily stained than the nucleoplasm. Possible explanations for this apparent anomaly are considered. It was concluded that DNA diffused out of the nucleus during embedding and sectioning, and that the colouration of the nucleolus and cytoplasm with the eight staining systems used was due to other nucleotides present.     

Fibrillar nucleolar remnants do not contain macromolecular precursors of ribosomal RNA : Demonstration by the effects of -galactosamine

Administration of -galactosamine to rats produces inhibition of liver nuclear RNA synthesis and associated alterations in the structure of the nucleolus. Polyacrylamide gel electrophoretic analysis of liver nuclear RNA from galactosamine-treated rats has shown the virtual complete absence of ribosomal RNA (rRNA) precursor molecules at a time when the nucleolus consists solely of a dense fibrillar core devoid of granules. No evidence for an artefactual, preferential breakdown of nuclear RNA during extraction could be obtained from either 2.7 or 8% acrylamide gels. Furthermore, the almost complete cessation of nuclear RNA synthesis makes the possibility of there being rapid synthesis and degradation of ribosomal precursor molecules in vivo unlikely. With toluidine blue stains for RNA with nuclei isolated from galactosamine-treated animals, the large, brightly staining area associated with the normal nucleolus was not seen. On the basis of these observations, it is concluded that an RNA-depleted nucleolus appears fibrillar. It is suggested that the fibrillar material of a normal nucleolus may not itself be RNA even though this region does contain RNA precursor molecules.     

Intranucleolar localization of the RNA polymerase A activity in isolated nuclei of regenerating rat liver

Ultrastructural autoradiography was used to visualize RNA polymerase A activity in parenchymal cell nuclei isolated from normal and regenerating (3, 24, 36 and 48 h after partial hepatectomy) rat liver. High resolution autoradiography showed that the activity of RNA polymerase A which was not inhibited by α-amanitin in a concentration of 0.8 μg/ml, was restricted to the nucleolus. Both the distribution pattern and number of grains were similar in control liver and regenerating liver 3 h after hepatectomy. Twentyfour, 36, and 48 h after hepatectomy nucleoli were enlarged and labeling was distinctly increased. In all experimental groups the activity of RNA polymerase A was located within fibrillar components of the nucleolus. The association of enzyme activity with this component was especially distinct in later stages (36 and 48 h) of liver regeneration. These results suggest that the fibrillar component of the nucleolus contains the active template for ribosomal RNA (rRNA) synthesis in rat liver cell nuclei.     

Ultrastructural localization of nucleic acid sequences in Saccharomyces cerevisiae nucleoli

The putative nucleolus in Saccharomyces cerevisiae is visible in electron micrographs as a darkly stained, crescent-shaped structure associated with the nuclear envelope. The haploid yeast genome contains 100 200 tandem copies of a 9.1 kb ribosomal DNA (rDNA) repeat predicted to reside in this structure. We combined in situ hybridization of non-isotopically labeled probes to isolated S. cerevisiae nuclei with immunogold detection to localize rDNA and rRNA precursor sequences in nuclei at the electron microscope (EM) level. Gold particles are restricted to defined regions of nuclei which appear more electron dense than the bulk of the nucleus and which generally exhibit the crescent shape typical of the structure thought to be the nucleolus. In addition, snR17, the yeast homolog of mammalian U3, a nucleolar-restricted small nuclear RNA (snRNA), was localized to the same electron dense region of the nucleus. These data, in conjunction with published immunofluorescent localizations of nucleolarassociated antigens, provide definitive proof that the dense crescent is the nucleolus. Finally, the technique described is applicable to probing nuclear organization in a genetically manipulable system.Abbreviations snRNA small nuclear RNA - AAF N-acetoxy-2-acetyl-aminofluorence by M.L. Pardue     

Effect of the ionic environment on the transcriptional activity of rat liver nucleoli

The localization of various RNA polymerase activities in the nucleoplasm and the nucleolus was investigated in isolated nuclei and in isolated nucleoli by means of a combination of cell fractionation, biochemical analysis and ultrastructural autoradiography. This resulted in four chief conclusions.

1. 1. The two main RNA polymerase activities can be localized clearly in the interphase nucleus: at low ionic strength, the activity is restricted to the nucleolus whereas at high ionic strength the activity is found in both nucleolar and nucleoplasmic regions.

2. 2. The preservation of the ultrastructure of the nuclei at high ionic strength is rather poor so that the physiological meaning of the activity revealed is questionable.

3. 3. Contrary to other reports, the nucleolar activity is significantly enhanced by increasing ammonium sulphate concentration in the presence of Mn²⁺.

4. 4. This increase is probably related to progressive revealing of RNA polymerase B activity within the nucleolus.

     

Some chemical properties of isolated pea nucleoli

Isolated nuclei and nucleoli of ungerminated pea embryos have been analyzed chemically for their content of DNA, RNA, zinc, iron, phosphorus, and protein sulfhydryl groups. The values obtained cannot be considered to represent the whole of the living nucleolar body as an undetermined amount of material is extracted from nucleoli in the course of their isolation. Only negligible amounts of DNA have been found in the isolated nucleoli; most of the DNA released on disruption of nuclei appears in a fraction showing very few structures under the light microscope. RNA is more concentrated in the nucleolus than in the nucleus or cytoplasm, but since nucleolar protein is 6 per cent of nuclear and less than 1 per cent of cytoplasmic protein, the total amount of nucleolar RNA is comparatively small. None of the other components listed occurs in high concentration in either nucleus or nucleolus.     

Some Chemical Properties of Isolated Pea Nucleoli

Isolated nuclei and nucleoli of ungerminated pea embryos have been analyzed chemically for their content of DNA, RNA, zinc, iron, phosphorus, and protein sulfhydryl groups. The values obtained cannot be considered to represent the whole of the living nucleolar body as an undetermined amount of material is extracted from nucleoli in the course of their isolation. Only negligible amounts of DNA have been found in the isolated nucleoli; most of the DNA released on disruption of nuclei appears in a fraction showing very few structures under the light microscope. RNA is more concentrated in the nucleolus than in the nucleus or cytoplasm, but since nucleolar protein is 6 per cent of nuclear and less than 1 per cent of cytoplasmic protein, the total amount of nucleolar RNA is comparatively small. None of the other components listed occurs in high concentration in either nucleus or nucleolus.