Fractionation of the nuclear matrix. I. Partial separation into matrix protein fibrils and a residual ribonucleoprotein fraction

Isolated rat liver nuclear matrices have been partially separated by means of mild sonication into a matrix protein (matricin) fraction and a residual ribonucleoprotein (RNP) fraction. The initial matricin fraction is composed largely of protein (91.1%) but also contains significant amounts of DNA (8.4%). Reconstruction experiments indicate that this DNA is not the result of the artifactual binding of DNA to the matrix during the extraction procedures. Subsequent treatment with DNase I results in purified matricin composed of greater than 99.5% protein. SDS acrylamide gel electrophoresis of the matrix protein fibrils reveals only three bands: the primary matrix polypeptides of 62,000, 66,000, and 70,000 daltons. Electron microscopy demonstrates a diffuse reticulum with fibrils as thin as 30--50 A and the presence of 80--100-A globular structures. The residual RNP fraction is composed largely of protein (80.1%) and RNA (19.5%), with only traces of DNA (1.1%). Over 98% of the total matrix-associated RNA is recovered in this fraction. SDS acrylamide gel electrophoresis indicates an enrichment in both low and high molecular weight secondary matrix polypeptides, although the 60,000--70,000-dalton polypeptides are present in significant amounts as well. Ultrastructural analysis of the residual RNP fraction reveals distinct electron-dense-staining matrix particles (150--350 A) attached to a fibrous matricin network.     

Absolute stereochemistry of guaianolides, of matricin and its epimers, of yarrow proazulenes, and of chamazulene carboxylic acid

Known determinations of the absolute configuration of guaianolides were collected and found to be few. The absolute configurations of guaianolides rest on the assumption that 7-H always has alpha-orientation. For matricin, only the relative configuration was determined. On the basis of a detailed study of the NMR spectra of matricin and its epimers, and of synthetic, NMR, and CD studies with its decomposition product, chamazulene carboxylic acid, we were able to reconfirm the accepted 3S,3aR,4S,9R,9aS,9bS configuration of matricin.     

The stereochemistry of matricin and 4-epimatricin,proazulene sesquiterpene lactones from Artemisia arborescens

An investigation of Artemisia arborescens afforded, in addition to the known compounds matricin, artabsin and artemetin, the new guaianolide 4-epimatricin. The stereostructures of 4-epimatricin and matricin were assigned on the basis of spectroscopic evidence.     

Nuclear matrix: isolation and characterization of a framework structure from rat liver nuclei

A nuclear framework structure termed the nuclear matrix has been isolated and characterized. This matrix forms the major residual structure of isolated nuclei and consists largely of protein with smaller amounts of RNA, DNA, carbohydrate, and phospholipid. The nuclear matrix can be further resolved by combined treatment with DNase and RNase. The remaining nuclear protein structure, after extraction of 90 percent of the nuclear protein, 99.9 percent of the DNA, and 98 percent of the RNA and phospholipid, is termed the nuclear protein matrix. Electron microscopy of this final nuclear protein matrix reveals an interior framework structure composed of residual nucleolar structures associated with a granular and fibrous internal matrix structure. The internal matrix framework is derived from the interchromatinic structures of the nucleus, and is connected to a surrounding residual nuclear envelope layer containing residual nuclear pore complex structures. Sodium dodecyl sulfate-acrylamide gel electrophoresis of the nuclear matrix proteins demonstrates three major polypeptide fractions, P-1, P-2, and P-3, with average molecular weights of approximately 69,000, 66,000 and 62,000, as well as several minor polypeptides which migrate at approximately 50,000 and at higher molecular weights (>100,000). Polypeptides with molecular weights identical to those of P-1, P-2 and P-3 are also components of isolated nuclear envelopes and nucleoli, whereas isolated chromatin contains no detectable matrix polypeptides. This suggests that the major matrix polypeptides are localized in specific structural regions of the nucleus, i.e., nuclear envelope, nucleoli, and interchromatinic structures. The presence of cytochrome oxidase activity in the isolated nuclear matrix indicates that at least some integral proteins of the nuclear membrane are associated with the matrix.     

Nuclear envelope and nuclear pore complex structure and organization in tobacco BY-2 cells

The nuclear envelope (NE) is a fundamental structure of eukaryotic cells with a dual role: it separates two distinct compartments, and enables communication between them via nuclear pore complexes (NPCs). Little is known about NPCs and NE structural organization in plants. We investigated the structure of NPCs from both sides of the NE in tobacco BY-2 cells. We detected structural differences between the NPCs of dividing and quiescent nuclei. Importantly, we also traced the organizational pattern of the NPCs, and observed non-random NPC distribution over the nuclear surface. Lastly, we observed an organized filamentous protein structure that underlies the inner nuclear membrane, and interconnects NPCs. The results are discussed within the context of the current understanding of NE structure and function in higher eukaryotes.     

Nuclear Pore Complex Structure in Birds

The nuclear envelope consists of two parallel membranes enclosing an aqueous lumen. In places there are pores in both membranes at which the two membranes are joined. Within these pores reside the nuclear pore complexes. The current structural models of the nuclear pore complex have been derived from a number of studies using different electron microscopical techniques. Recently, using surface imaging techniques such as field emission in-lens scanning electron microscopy, novel structures have been identified, particularly at the periphery of the structure, most notably the nucleoplasmic basket. One limitation of the current models is that they are based almost entirely on nuclear envelopes isolated from amphibian oocytes and a pressing question is whether this structure is the same in other organisms and tissues. Here we have studied the structure of nuclear envelopes isolated from bird oocytes. We show that the overall structure is remarkably conserved. In particular, recently discovered peripheral structures appear very similar. We see variations in basket conformation but believe that this is related to the functional states of individual pore complexes.     

Revisiting the planarity of nucleic acid bases: Pyramidilization at glycosidic nitrogen in purine bases is modulated by orientation of glycosidic torsion

We describe a novel, fundamental property of nucleobase structure, namely, pyramidilization at the N1/9 sites of purine and pyrimidine bases. Through a combined analyses of ultra-high-resolution X-ray structures of both oligonucleotides extracted from the Nucleic Acid Database and isolated nucleotides and nucleosides from the Cambridge Structural Database, together with a series of quantum chemical calculations, molecular dynamics (MD) simulations, and published solution nuclear magnetic resonance (NMR) data, we show that pyramidilization at the glycosidic nitrogen is an intrinsic property. This property is common to isolated nucleosides and nucleotides as well as oligonucleotides—it is also common to both RNA and DNA. Our analysis suggests that pyramidilization at N1/9 sites depends in a systematic way on the local structure of the nucleoside. Of note, the pyramidilization undergoes stereo-inversion upon reorientation of the glycosidic bond. The extent of the pyramidilization is further modulated by the conformation of the sugar ring. The observed pyramidilization is more pronounced for purine bases, while for pyrimidines it is negligible. We discuss how the assumption of nucleic acid base planarity can lead to systematic errors in determining the conformation of nucleotides from experimental data and from unconstrained MD simulations.     

A modified procedure for the isolation of a pore complex-lamina fraction from rat liver nuclei

A modified procedure for the isolation of a nuclear pore complex-lamina fraction from rat liver nuclei is described. Evidence is provided that the isolated lamina, a 150-A thick, proteinaceous structure, apposes the inner nuclear envelope membrane, connecting nuclear pore complexes and surrounding the entire nucleus.     

Isolated Plant Nuclei Nucleate Microtubule Assembly: The Nuclear Surface in Higher Plants Has Centrosome-like Activity

In most eukaryotic cells, microtubules (MTs) are assembled at identified nucleating sites, such as centrosomes or spindle pole bodies. Higher plant cells do not possess such centrosome-like structures. Thus, the fundamental issues of where and how the intracellular plant MTs are nucleated remain highly debatable. A large body of evidence indicates that plant MTs emerge from the nuclear periphery. In this study, we developed an in vitro assay in which isolated maize nuclei nucleate MT assembly at a tubulin concentration (14 [mu]M of neurotubulin) that is not efficient for spontaneous MT assembly. No MT-stabilizing agents, such as taxol or dimethyl sulfoxide, were used. Our model provides evidence that the nuclear surface functions as a MT-nucleating site in higher plant cells. A monoclonal antibody raised against a pericentriolar antigen immunostained the surface of isolated nuclei, and a 100-kD polypeptide in 4 M urea-treated nuclear extracts was detected.     

Assessing genetic structure with multiple classes of molecular markers: a case study involving the introduced fire ant Solenopsis invicta.

We used 30 genetic markers of 6 different classes to describe hierarchical genetic structure in introduced populations of the fire ant Solenopsis invicta. These included four classes of presumably neutral nuclear loci (allozymes, codominant random amplified polymorphic DNAs (RAPDs), microsatellites, and dominant RAPDs), a class comprising two linked protein-coding nuclear loci under selection, and a marker of the mitochondrial DNA (mtDNA). Patterns of structure revealed by F statistics and exact tests of differentiation were highly concordant among the four classes of neutral nuclear markers, although the microsatellites were the most effective markers for detecting structure. The results from the mtDNA complemented those from the neutral nuclear markers by revealing that strong limitations to female-mediated gene flow were the cause of the local structure registered by the nuclear markers. The pattern of structure inferred from the selected nuclear loci was markedly different from the patterns derived from the other sets of markers but was predictable on the basis of the presumed mode of selection acting on these loci. In general, the results for all six classes of markers can be explained by known features of the social and reproductive biology of fire ants. Thus, the results from these diverse sets of markers, combined with detailed natural history data, provide an unusually complete picture of how the fundamental evolutionary forces of gene flow, drift, and selection govern the distribution of genetic variation within and between fire ant populations.     

The polypeptide composition and ultrastructure of nuclear ghosts isolated from mammalian cells

The polypeptide species of non-membranous nuclear ghosts from purified cell nuclei are conserved among a variety of human, hamster and mouse cell types studied, including HeLa, BHK, 3T6 and Hep-2 cell lines. The polypeptide species present in nuclear ghosts from HeLa cells synchronized in various stages of the cell cycle are largely the same with minor variations. The isolated nuclear ghosts are similar, in terms of polypeptide composition, to other residual nuclear structures isolated by independent techniques. The nuclear ghosts appear as flattened sac-like structures when viewed by scanning electron microscopy. Transmission electron microscopy of the nuclear ghosts reveals ring-like structures which may represent the nuclear pores. Also observed are novel rod-shaped structures approximately 260 nm in length and 50 nm in diameter. The latter images either arise by a rearrangement during isolation of the nuclear ghost macromolecules or are a heretofore undescribed structure of intact nuclei.     

The major polypeptides of the nuclear pore complex

Nuclear envelopes of maturing oocytes of various amphibia contain an unusually high number of pore complexes in very close packing. Consequently, nuclear envelopes, which can be manually isolated in great purity, provide a remarkable enrichment of nuclear pore complex material, relative to membranous and other interporous structures. When the polypeptides of nuclear envelopes isolated from oocytes of Xenopus laevis and Triturus alpestris are examined by gel electrophoresis, visualized either by staining with Coomassie blue or by radiofluorography after in vitro reaction with [³H]dansyl chloride, a characteristic pattern is obtained (10 major and 15 minor bands). This polypeptide pattern is radically different from that of the nuclear contents isolated from the same cell. Extraction of the nuclear envelope with high salt concentrations and moderately active detergents such as Triton X-100 results in the removal of membrane material but leaves most of the non-membranous structure of the pore complexes. The dry weight of the pore complex (about 0.2 femtograms) remains essentially unchanged during such extractions as measured by quantitative electron microscopy. The extracted preparations which are highly enriched in nuclear pore complex material contain only two major polypeptide components with apparent molecular weights of 150 000 and 73 000. Components of such an electrophoretic mobility are not present as major bands, if at all, in nuclear contents extracted in the same way. It is concluded that these two polypeptides are the major constituent protein(s) of the oocyte nuclear pore complex and are specific for this structure. When nuclear envelopes are isolated from rat liver and extracted with high salt buffers and Triton X-100 similar bands are predominant, but two additional major components of molecular weights of 78 000 and 66 000 are also recognized. When the rat liver nuclear membranes are further subfractionated material enriched in the 66 000 molecular weight component can be separated from the membrane material, indicating that this is relatively loosely associated material, probably a part of the nuclear matrix. The results suggest that the nuclear pore complex is not only a characteristic ubiquitous structure but also contains similar, if not identical, skeletal proteins that are remarkably resistant to drastic changes of ionic strength as well as to treatments with detergents and thiol reagents.     

Characterization of lamina-bound chromatin in the nuclear shell isolated from HeLa cells

The structure and the polypeptide composition of the nuclear shell isolated from interphase HeLa cells have been investigated and compared to those of the intranuclear material. The isolated nuclear shell contains chromatin superstructures (28-32 nm thick fibres) made of tightly packed nucleosomes that resist low ionic strength conditions and that are associated with the three nuclear lamins. Chromatin in the nuclear shell exhibits very simple chemical composition. Especially, non-histone proteins are lacking. The results presented here rule out the possibility that the nuclear shell results from contamination of lamina by intranuclear elements. They suggest that the lamins are directly involved in the specific properties and in the organization of chromatin in the nuclear shell.     

Population structure of <Emphasis Type="Italic">Apis cerana</Emphasis> in Thailand reflects biogeography and current gene flow rather than <Emphasis Type="Italic">Varroa</Emphasis> mite association

Concordance between the mitochondrial haplotypes of the Eastern honey bee, Apis cerana, and its ectoparasitic Varroa mites across the Isthmus of Kra in Thailand has suggested that local host–pathogen co-evolution may be responsible for the geographic distribution of particular genotypes. To investigate nuclear microsatellites population structure in A. cerana, single workers of A. cerana colonies from Thailand were genotyped at 18 microsatellite loci. The loci showed intermediate to high levels of heterozygosity and a range of allele numbers. The analyses confirmed a fundamental subdivision of the Thai A. cerana population into the “Asia Mainland” and “Sundaland” regions at the Isthmus of Kra. However, the nuclear microsatellite differentiation was less distinct than mtDNA haplotype differences, suggesting male-biased dispersal and population admixture. Overall, samples showed a weak isolation-by-distance effect. The isolated population on Samui island was most differentiated from the other samples. The results do not support our initial hypothesis of local host–pathogen co-evolution, which predicts a strict correspondence between the nuclear genome and the lineage of parasitic Varroa mite of the A. cerana samples, because the gene flow indicated by our nuclear microsatellite markers should also mix potential Varroa resistance alleles among subpopulations. Instead, our study suggests that the coincidental distribution of Varroa lineages and A. cerana population structure in Thailand are the result of biogeographic history and current migration patterns.     

Identification of a sperm nuclear annulus: a sperm DNA anchor

We report the isolation and characterization of a new nuclear structure from spermatozoa of the golden Syrian hamster which we term the nuclear annulus. The nuclear annulus was located at the implantation fossa, the point at which the tail is joined to the sperm head, inside the nucleus adjacent to the inner nuclear envelope. Extraction of sperm nuclei with 2 M NaCl and 10 mM dithiothreitol caused the solubilization of the protamines and DNA decondensation. This released the DNA from its structural constraints except that the DNA remained anchored to the nuclear annulus, which survived the extraction procedure. Nuclear annuli were isolated and examined by scanning electron microscopy. The nuclear annulus was a double-crescent, ring-shaped structure, 2.10 +/- 0.16 micron long and 1.36 +/- 0.13 micron wide. We believe that the nuclear annulus may play an important role in the organization of sperm DNA.     

Isolation and partial characterization of the nuclear shell of HeLa cells

We recently described the spontaneous dissociation from isolated HeLa cell chromatin of a nuclear shell consisting of the outermost layer of peripheral chromatin and of fibrous lamina material [1–3]. The present paper reports our attempts to isolate and purify this chromatin subfraction. Preliminary results of electron microscopy and sucrose gradient centrifugation indicate that nuclear shell DNA accounts for 0.8–1% of total nuclear DNA, and is presumably constrained in highly stable supranucleosomes by a specific protein environment. Small variable amounts of RNA are also found in the nuclear shells but do not seem to be involved in maintaining shell structure. Further studies of the isolated nuclear shells should shed some light on the specific organization of the chromatin adjacent to the inner nuclear membrane in the nucleus in situ.