蚕豆细胞核仁和核质中剪接因子SC35的定位研究

SC35 is a non-snRNP spliceosome component purified from mammalian cells by Fu and Maniatis in 1990. In vitro splicing assays showed that SC35 plays a key role in splicing site selection and ATP-dependent pre-spliceosome assembly. In the mammalian nucleus, SC35 has been localized to distinct and dynamic nuclear domains: immunofluorescence observations revealed the presence of SC35 in speckles distributed in various regions throughout the nucleoplasm, which, as identified with immunoelectron microscopy, correspond to the interchromatin granules (IGs) and perichromatin fibrils (PFs). However, there has been no report regarding the presence and distribution pattern of SC35 in higher plant nuclei. Engage in such studies will surely contribute to our understanding of RNA processing and the spatial organization or structure basis of this process in higher plant. In this article, we studied the distribution pattern of SC35 in the nucleus of the root meristematic cells of Vicia faba by immunoelectron microscopy. After immunolabeling with anti-SC35 mAb and protein A-colloidal gold, IGs and PFs in the nucleoplasm and dense fibrillar component (DFC) of the nucleolus were heavily labeled with gold particles, while only a few of the gold particles were found in fibrillar centers (FC) and nucleolar vacuoles (NV) of the nucleolus and the central domains of the condensed chromatin. Densities of gold particles in the areas of DFC and the area of IGs plus PFs were 65.89/microns 2 and 36.28/microns 2 respectively, much higher than that of the central domain of condensed chromatin and that of FC plus NV, which were only 5.90/microns 2 and 6.26/microns 2 respectively. This indicates that DFC of the nucleolus and the area of IGs plus PFs of the nucleoplasm are enriched with SC35 or SC35-like protein. The distribution pattern of SC35 or SC35-like protein in the nucleoplasm of Vicia faba is similar to that of the mammalian nuclei. To the authors' knowledge, it is a new finding that SC35 or SC35-like protein exists in the nucleolus.     

Ultrastructural localization of DNA and RNA in Allium porrum interphase cells by means of nuclease-gold complexes

Summary Nucleic acids have been localized inAllium porrum interphase meristematic cells by means of labelling with nuclease-gold complexes, a technique which provides high resolution and improved specificity. DNase-gold labelling was observed over dense chromatin and to a lesser extent over dispersed chromatin. Nucleolar labelling was restricted to the dense fibrillar component, very few particles being located over the fibrillar centres. Labelling by the RNase-gold complex was present over both the cytoplasm and the nucleoplasm. Cytoplasm labelling was intense over the rough endoplasmic reticulum but absent over vacuoles. In the nucleoplasm many gold particles were located at the border between the condensed and the dispersed chromatin. Nucleolar labelling was intense over the granular zones but many gold particles were also seen over the dense fibrillar component. Fibrillar centres showed, however, no labelling with the RNase-gold complex. These results are consistent with previous autoradiographic and cytochemical observations carried out on the same plant material.     

High Mobility Group 1 Protein Is Not Stably Associated with the Chromosomes of Somatic Cells

High mobility group 1 (HMG1) protein is an abundant and conserved component of vertebrate nuclei and has been proposed to play a structural role in chromatin organization, possibly similar to that of histone H1. However, a high abundance of HMG1 had also been reported in the cytoplasm and on the surface of mammalian cells. We conclusively show that HMG1 is a nuclear protein, since several different anti-HMG1 antibodies stain the nucleoplasm of cultured cells, and epitope-tagged HMG1 is localized in the nucleus only. The protein is excluded from nucleoli and is not associated to specific nuclear structures but rather appears to be uniformly distributed. HMG1 can bind in vitro to reconstituted core nucleosomes but is not stably associated to chromatin in live cells. At metaphase, HMG1 is detached from condensed chromosomes, contrary to histone H1. During interphase, HMG1 readily diffuses out of nuclei after permeabilization of the nuclear membranes with detergents, whereas histone H1 remains associated to chromatin. These properties exclude a shared function for HMG1 and H1 in differentiated cells, in spite of their similar biochemical properties. HMG1 may be stably associated only to a very minor population of nucleosomes or may interact transiently with nucleosomes during dynamic processes of chromatin remodeling.     

Ultrastructural studies of spermatogenesis in the anthocerotales. III. Gamete morphogenesis: From spermatogenous cell through midstage spermatid

An ultrastructural examination of spermatogenesis in Phaeoceros has shown nucleoli to be present in spermatogenous cells and to persist until the centrioles become associated with nuclei of young spermatids. At the onset of multilayered structure (MLS) formation, well-defined aggregations of osmiophilic strands begin to form in the nuclei of young spermatids and disappear shortly after chromatin condensation starts in the midstage spermatids. When the centrioles in the young spermatids are orientated perpendicular to the nuclear envelope, the nucleoplasm immediately in front of them is densely stained. Where the spline tubules of the MLS extend over the nucleus, the nuclear envelope is devoid of pores, and the inner nuclear membrane is contacted internally by the local deposition of dense staining nucleoplasm. Chromatin condensation begins with strands extending perpendicularly from the dense staining nucleoplasm beneath the spline and continues with the nuclear beak becoming filled with condensed chromatin. As the MLS lamellae disappear acropetally, the rear portion of the anterior mitochondrion (AM) extends back under the nuclear beak which now narrows to a size that approximates the anterior end of the nucleus of a spermatozoid. By the end of the mid-spermatid stage, the nucleus has coiled approximately one gyre of a helix and the five or six central slpine tubules extend over the plastid which is now located beneath the front end of the AM. Several profiles of endoplasmic reticulum confluent with the nuclear envelope are present. Possible factors which might play a role in determining the morphology of the mid-spermatids are discussed.     

Fine structural in situ analysis of nascent DNA movement following DNA replication

Nascent DNA (newly replicated DNA) was visualized in situ with regard to the position of the previously replicated DNA and to chromatin structure. Localization of nascent DNA at the replication sites can be achieved through pulse labeling of cells with labeled DNA precursors during very short periods of time. We were able to label V79 Chinese Hamster cells for as shortly as 2 min with BrdU; Br-DNA, detected by immunoelectron microscopy, occurs at the periphery of dense chromatin, at individual dispersed chromatin fibers, and within dispersed chromatin areas. In these regions DNA polymerase alpha was also visualized. After a 5-min BrdU pulse, condensed chromatin also became labeled. When the pulse was followed by a chase, a larger number of gold particles occurred on condensed chromatin. Double-labeling experiments, consisting in first incubating cells with IdU for 20 min, chased for 10 min and then labeled for 5 min with CldU, reveal CldU-labeled nascent DNA on the periphery of condensed chromatin, while previously replicated IdU-labeled DNA has been internalized into condensed chromatin. Altogether, these results show that the sites of DNA replication correspond essentially to perichromatin regions and that the newly replicated DNA moves rapidly from replication sites toward the interior of condensed chromatin areas.     

两种蝇类精细胞发育过程中细胞核的变态

本文用透射电子显微镜研究了大头金蝇(hrysomyia megacephala)和肥须亚麻蝇(Parasarcophaga crassipalpis)精细胞发育过程中细胞核的变态过程.精细胞从球形细胞演变为线形精子,核要经历四个时期,即:球核期,细胞为球形,核亦为球形,核膜与一般体细胞核无异;棒核期,核拉长如棒,顶体形成,核膜孔聚集于一侧;染色质凝聚期,染色质与核质分开,经过一系列变化,再凝聚成致密的块状,多余核质从核孔聚集处开口排出核外;成熟期,核变成一团电子密度极大的腊肠形.精细胞抛弃绝大部分细胞质和多余的结构,变成线形精子.以上演变过程两种蝇类完全相似,但在染色质凝聚期的变化中差异却很大:大头金蝇凝聚程序为:细纤维—粗纤维—块状—致密团;肥须亚麻蝇则为:蚁蚕状—纵列薄片状—厚片状—块伙—致密团.     

Fine Structural in Situ Analysis of Nascent DNA Movement Following DNA Replication

Nascent DNA (newly replicated DNA) was visualized in situ with regard to the position of the previously replicated DNA and to chromatin structure. Localization of nascent DNA at the replication sites can be achieved through pulse labeling of cells with labeled DNA precursors during very short periods of time. We were able to label V79 Chinese Hamster cells for as shortly as 2 min with BrdU; Br-DNA, detected by immunoelectron microscopy, occurs at the periphery of dense chromatin, at individual dispersed chromatin fibers, and within dispersed chromatin areas. In these regions DNA polymerase α was also visualized. After a 5-min BrdU pulse, condensed chromatin also became labeled. When the pulse was followed by a chase, a larger number of gold particles occurred on condensed chromatin. Double-labeling experiments, consisting in first incubating cells with IdU for 20 min, chased for 10 min and then labeled for 5 min with CldU, reveal CldU-labeled nascent DNA on the periphery of condensed chromatin, while previously replicated IdU-labeled DNA has been internalized into condensed chromatin. Altogether, these results show that the sites of DNA replication correspond essentially to perichromatin regions and that the newly replicated DNA moves rapidly from replication sites toward the interior of condensed chromatin areas.     

Distribution of sugar-binding sites within interphase nuclei and mitotic chromosomes of a human cell line

Using gold labelled neoglycoproteins containing either alpha-D-glucose, N-acetyl-beta-D-glucosamine, alpha-D-mannose, 6-phospho-alpha-D-mannose, and alpha-L-fucose (BSA), we investigated their intranuclear binding sites in the TG human cell line. Although gold-labelled BSA did not give any noticeable labelling, the presence of 1% free BSA in the medium containing the gold labelled neoglycoproteins was revealed to be a key factor of the labelling. During interphase in the presence of free BSA most of the labelling was detected in the nucleoplasm. The border of the condensed chromatin, known to be the site of hnRNA synthesis as well as the interchromatin areas enriched in RNPs were labelled. Condensed chromatin also contained binding-sites. The nucleolus was seen to present low labelling in comparison with the labelling observed over the nucleoplasm. These nucleolar binding sites were located both in the dense fibrillar and granular components. No labelling could be detected over the fibrillar centers which are very conspicuous in this cell line. During mitosis sugar-binding sites were observed over the chromosomes. Data reported here show for the first time that lectin-like proteins and chromatin components are colocalized both during interphase and mitosis. In addition, within the nucleolus the presence of sugar-binding proteins was seen to be restricted to the dense fibrillar and granular components.     

Immunoelectron microscopic localization of progesterone receptor in the chick oviduct

A peroxidase-anti-peroxidase (PAP) method using polyclonal anti-PR antibodies was used to localize progesterone receptor (PR) electron microscopically in the chick oviduct. The immunoreaction precipitate indicating PR was localized inside the nuclei of epithelial, glandular and stromal cells. In the estrogen withdrawn oviduct cytoplasmic immunoreaction precipitate was not seen. Inside the nucleus unoccupied PR was localized mainly like the heterochromatin. As visualized by the PAP technique, the localization of PR was not systematically changed after progesterone administration. In conclusion, we suggest that progesterone receptor in the chick oviduct is an intranuclear protein.     

Immunoelectron-microscopic localization of a proliferation-associated antigen Ki-67 in MCF-7 cells

Summary Immunocytochemistry using the monoclonal antibody Ki-67 is a commonly used method to assess proliferative activity of malignant tumours. Ki-67 reacts with proliferating cells with an antigen, whose structure, function and exact locations are unknown. We studed the subcellular location of Ki-67 in MCF-7 cells using immunoelectron microscopy. In the interphase cells, Ki-67 immunoreactivity was localized in the nucleolus, mainly in the nucleolar cortex. In particular areas of the granular component of the nucleolus were strongly stained. Weak spot-like nucleoplasmic immunostaining was also seen outside the nucleolus. During prophase Ki-67 antigen was localized on the surfaces of the condensed chromatin and during metaphase on the surface of the chromosomes. After cell division and prior to formation of new nucleoli, Ki-67 immunoreactivity was located in the nucleoplasm. Quantification of Ki-67 immunofluorescence signal by flow cytometry revealed highest Ki-67 levels in mitotic cells. The localtion of Ki-67 is very similar to certain recently described proteins of nucleolar preribosomes suggesting that Ki-67 may also be a component of the preribosomes.     

PTP-S2, a nuclear tyrosine phosphatase, is phosphorylated and excluded from condensed chromosomes during mitosis

PTP-S2 is a tyrosine specific protein phosphatase that binds to DNA and is localized to the nucleus in association with chromatin. It plays a role in the regulation of cell proliferation. Here we show that the subcellular distribution of this protein changes during cell division. While PTP-S2 was localized exclusively to the nucleus in interphase cells, during metaphase and anaphase it was distributed throughout the cytoplasm and excluded from condensed chromosomes. At telophase PTP-S2 began to associate with chromosomes and at cytokinesis it was associated with chromatin in the newly formed nucleus. It was hyperphosphorylated and showed retarded mobility in cells arrested in metaphase. In vitro experiments showed that it was phosphorylated by CK2 resulting in mobility shift. Using a deletion mutant we found that CK2 phosphorylated PTP-S2 in the C-terminal non-catalytic domain. A heparin sensitive kinase from mitotic cell extracts phosphorylated PTP-S2 resulting in mobility shift. These results are consistent with the suggestion that during metaphase PTP-S2 is phosphorylated (possibly by CK2 or a CK2-like enzyme), resulting in its dissociation from chromatin.     

The effect of progesterone on the localization of progesterone receptors in the nuclei of chick oviduct cells

Summary The location of occupied and unoccupied progesterone receptors (PR) in chick oviduct cells was studied by immuno-electron microscopy with the use of a highly specific polyclonal anti-PR antibody and pre-embedding modifications of the peroxidase-anti-peroxidase-(PAP-) or immunogold-silver methods. Both methods revealed a nuclear localization of the PRs. The location of the PR in the nucleus was studied in detail by means of the immunogold-silver method. The most intense labelling for unoccupied PRs was in the condensed chromatin. After occupation of PRs with progesterone (P), decondensation or dispersion of chromatin was observed. At the same time, the labelling in the border area of condensed and dispersed chromatin, and in the dispersed chromatin, increased. The changes were statistically significant. The results can be explained by conformational changes of the PR-containing chromatin rather than by translocation of PRs from one site to another.     

Fine structure of the germinating cells during the spermiogenesis of Arion rufus (Mollusca,Pulmonate Gastropoda)

Changes in spermatozoan ultrastructure have been studied during spermiogenesis of the slug Arion rufus (Gastropoda, Pulmonata, Stylommatophora). The ovotestis was investigated during the male stage, definite by the presence of spermatozoa. Some peculiar characteristics are shown by early spermatids: Around the nucleus, the nuclear envelope presents two thick layers located on opposite sides, the apical and basal plates, that will determine the antero-posterior axis of the spermatid. The chromatin, first dispersed throughout the nucleoplasm gives later on thick filaments which become attached over the inner surface of these plates. The chromatin filaments are then arranged parallel to the antero-posterior axis as the nucleus elongates. The position of the plates determines the antero-posterior axis of the spermatid. In the mature spermatozoa, the chromatin is more condensed and the nucleus presents an helical organization. The acrosome and flagellum are respectively attached externally to the center of the apical and basal plates. The acrosome consists of a membrane-bound vesicle and forms a column of homogeneous material. In the middle piece, the mitochondria have been transformed into a mitochondrial derivate by the way of a complicated metamorphosis. The axoneme is surrounded by three mitochondrial helices but only one of them contains glycogene granules. © 1996 Wiley-Liss, Inc.     

Ultrastructural studies of H-1 parvovirus replication : III. Intracellular localization of viral antigens with immunocytochrome c

Immunoelectron microscopy with cytochrome c conjugated anti-H-1 IgG was used to localize antigens of the parvovirus H-1 within synchronized human NB cells. Since glutaraldehyde destroyed H-1 antigenicity, a fixative containing formaldehyde was developed which preserved both cellular ultrastructure and antigenic function. The earliest H-1 specific staining occurred on the heterochromatin bordering the nuclear envelope at 8 h post infection (p.i.). At 10 h p.i., labeling was found on the chromatin associated with the nucleolar surface and tufts of heterochromatin distributed throughout the nucleoplasm. Except for this H-1 labeling, the chromatin appeared indistinguishable from that of uninfected cells. By 12 h p.i., however, coinciding with the abrupt rise in synthesis of H-1 hemagglutinin and infectious virus, H-1 labeled intranuclear chromatin had condensed and migrated toward the nuclear membrane. Also, trabeculae of intranuclear chromatin were tagged with anti-H-1 conjugate as contraction of nucleolar chromatin and disintegration of nucleolar ultrastructure began. Condensation of the nucleolar associated chromatin and nuclear heterochromatin appeared complete by 18–36 h p.i. when thick zones of this H-1 labeled material were observed at the nucleolar and nuclear periphery. Our results indicate that the binding of unassembled H-1 proteins to specific regions of chromatin is associated with their condensation and margination, resulting in early nucleolar destruction and subsequent nuclear damage during H-1 infection.     

Restriction enzyme analysis of DNA methylation in "condensed" chromatin of Ha-ras-transformed NIH 3T3 cells.

Increased amounts of chromatin condensation (i.e., localized areas of high DNA density, or chromatin higher order packing state) have been described in NIH 3T3 cells transformed with the Ha-ras oncogene. The structural basis for this oncogene-mediated alteration in nuclear organization is unknown. Since DNA methylation is likely to be involved in regulating the nucleosomal level of DNA packaging, we studied the role of DNA methylation in higher-order chromatin organization induced by Ha-ras. CpG-methylated DNA content was estimated in "condensed" chromatin of Ha-ras-transformed NIH 3T3 cell lines which differ in ras expression and ras-induced metastatic ability but present approximately the same values of "condensed" chromatin areas. The question posed was that if DNA methylation were involved with the chromatin higher-order organization induced by Ha-ras in these cell lines, the methylated DNA density in the "condensed" chromatin would also be the same. The DNA evaluation was performed by video image analysis in Feulgen-stained cells previously subjected to treatment with Msp I and Hpa II restriction enzymes, which distinguish between methylated and non-methylated DNA. The amount of methylated CpG sequences not digested by Hpa II in "condensed" chromatin regions was found to vary in the studied ras-transformed cell lines. DNA CpG methylation status is thus suggested not to be involved with the higher order chromatin condensation induced by ras transformation in the mentioned NIH 3T3 cell lines.     

Pattern of DNA synthesis in the meristematic cells of sinapis

Summary High-resolution autoradiographs were made of ultrathin sections in the shoot apex and the very young leaves of Sinapis alba fed with tritiated thymidine for 4 hours. Three types of labeled nuclei were found. (1) Those labeled in both the dispersed and the condensed chromatin, (2) those labeled only in the dispersed chromatin, and (3) those labeled only in the condensed chromatin.A distinct cytoplasmic labeling was found. Proplastids and mitochondria were the only significantly labeled entities in the cytoplasm. DNA synthesis in these organelles seems to be synchronized with DNA synthesis in the nucleus.This work was carried out at the Department of Botany, University of California, Berkeley, during the tenure of a fellowship from I.R.S.I.A. (Institut pour l'Encouragement de la Recherche Scientifique dans l'Industrie et l'Agriculture), Belgium.