Molecular genetic analysis of the X-chromosomal nuclear envelope attachment region in nurse cells of the malaria mosquitoes Anopheles messeae Fall

DNA of the X-chromosomal nuclear envelope attachment region was isolated from malaria mosquito Anopheles messeae Fall. nurse cells by chromosome microdissection. A DNA library of the region was constructed using a plasmid vector. DNA sequencing revealed gene fragments, tandem repeats, and a great variety of transposable elements (TEs). The X-chromosomal nuclear envelope attachment region was concluded to correspond in molecular organization and cytogenetics to diffuse intercalary heterochromatin.     

Arrangement of chromosome ends and axial core formation during early meiotic prophase in the male grasshopper Brachystola magna by 3D,E.M. reconstruction

Evidence is presented that chromosome ends are attached to the nuclear envelope prior to the formation of axial cores during early meiotic prophase in the grasshopper Brachystola magna. The attachment sites of distal and proximal chromosome ends are clustered in a small region of the inner nuclear envelope resulting in a classical bouquet arrangement of the chromosomes. Proximal ends are tightly clustered due to the presence of chromocenters. Distal chromosome ends are more widely scattered throughout the base of the bouquet. —Axial core formation can be initiated at chromosome ends or at internal chromosome sites. However, there is a preference for axial cores to form in distal chromosome regions rather than proximal regions during early meiotic prophase. — Virtually all of the nuclear pore complexes are located in the general vicinity of the chromosome attachment sites but each specific attachment site is surrounded by a small area of nuclear envelope which is devoid of pore complexes.     

The fine structure of the parasitic dinoflagellateHaplozoon axiothellae

Summary The multicellular parasitic dinoflagellateHaplozoon axiothellae Siebert was studied with electron microscopy. The trophocyte (attachment cell) bears a suction apparatus with a movable protruding stylet that penetrates the epithelial cell of the host gut. The gonocytes are binucleate and divide frequently. Nuclear structure is similar to the mesokaryotic condition of other dinoflagellates although the chromosomes lack the helically coiled appearance of the DNA fibrils. During nuclear division the nucleus retains its envelope intact and cytoplasmic invaginations develop in which packets of parallel microtubules occur. The microtubules attach to the nuclear envelope opposite the site of chromosome attachment. The chromosomes remain condensed during interphase but the helically coiled DNA fibrils characteristic of the mesokaryotic condition are not evident.The theca which encloses all cells is composed of elements similar to those of typical free-living dinoflagellates, the outer cell membrane and flattened vesicles which contain either flat thin plates or larger spines. No subthecal microtubules are present. The theca grows inward following nuclear division and separates the daughter cells. Trichocysts, pusules, flagellar structures and chloroplasts are not present. The relationship ofHaplozoon to other free-living and parasitic dinoflagellates is discussed.     

Analysis of inactive X chromosome structure by in situ nick translation

Nick translation assays of fixed interphase female fibroblasts with tritiated nucleotides demonstrated a characteristic absence of label over sex chromatin. The chromatin bodies were nearly always peripheral in location and a ribbon of nick translatable DNA was detected between the sex chromatin and the nuclear envelope. High voltage electron microscopy indicated the possibility of a special nuclear envelope attachment region. The apparent resistance of sex chromatin to nick translation did not appear to be due to resistance to DNase I attack.     

Analysis of the spatial organization of the XL chromosome attachment site in nurse cell nuclei of the malaria mosquito <Emphasis Type="Italic">Anopheles atroparvus</Emphasis>

The spatial position of the site of XL chromosome attachment to the nuclear envelope of ovarian nurse cells relative to the oocyte has been analyzed in the malaria mosquito Anopheles atroparvus van Thiel. The XL chromosome attachment sites in the oocyte-nurse cell system of this species have been demonstrated to be orderly arranged, with the attachment sites in two out of three nurse cells in the same layer identically oriented relative to the oocyte.     

A freeze-fracture study of the nuclear envelope during spermiogenesis inMarsilea. Formation of a pore-free zone associated with the microtubule ribbon

Summary The nuclear envelope was freeze-fractured at different stages during spermiogenesis inMarsilea. Nuclear pores were seen at all stages of development, but were excluded from a specialized region of the nuclear envelope that is in contact with the microtubule ribbon. The microtubule ribbon is associated with one edge of the nuclear envelope throughout nuclear shaping and chromatin condensation and the pore-free zone may be a region of the nuclear envelope that is important in these processes.     

Fine structure of the macronucleus during the cell division cycle of Euplotes eurystomus,a Ciliate Protozoon

This paper reports new observations obtained from a study of macronuclear fine structure throughout various stages of the cell division cycle of Euplotes. Study of the ultrastructural organization of the macronuclear chromatin indicates that much of the chromatin is organized into continuous masses, portions of which appear to be attached to the nuclear envelope. The macronuclear envelope appears unchanged in the region of a replication band, and apparent attachments of the chromatin to the inner membrane of the nuclear envelope are maintained in the reticular and diffuse zones. Intranuclear helices were never observed in the diffuse zone. During macronuclear division, linear elements (fibrils or microtubules) were observed in close association with both chromatin bodies and nucleoli. The ultrastructural data suggest that the intranuclear linear fibrils have two functions: elongation of the dividing nucleus, and attachment of chromatin bodies and nucleoli to the envelope. The significance of these observations for macronuclear division and chromatin segregation is considered.     

Specific DNA sequences are associated with nuclear envelopes of pseudonurse cells in Drosophila melanogaster otu 11

Nuclei of ovarian pseudonurse cells from the mutant strain of Drosophila melanogaster otu 11 are suitable for mapping the attachment of chromosomes to the nuclear envelope (NE). Loci in contact with the NE included region 20CD of the X chromosome, region 41 of chromosome 2, the proximal end of region 81 of chromosome 3, and region 101 of chromosome 4. In situ hybridization revealed that all 4 regions contained sequences homologous to clone lambda20p1.4. DNA of clone lambda20p1.4 was previously found to bind specifically to purified D. melanogaster lamins. These results suggest that specific DNA sequences are involved in attachment of chromosomes to NE in vivo.     

Unusual nucleotide sequence of a DNA fragment isolated from nuclear envelopes of mouse hepatocytes

Fragments of chromosomal DNA isolated from nuclear envelopes of mouse hepatocytes were previously cloned and partially characterized in our laboratory. One of the cloned fragments (EnvM4) had unusual characteristics: abundant representability in the clone library (about 50%) and homology with DNA of archebacteria. An analysis of nucleotide sequence of this fragment conducted in more detail allowed the detection of an evolutionary conserved region present in the genomes of various organisms (from bacteria to human). Other characteristics of fragment EnvM4 suggest a possible functional role of chromosomal DNA regions determining the attachment of chromosomes to the nuclear envelope.     

On the nature of nuclear envelope-associated RNA

Nuclear envelopes were isolated from rat-liver nuclei. Nuclear envelope-associated RNA was isolated and hybridized to filter-bound DNA in the presence of competing RNA populations. Cytoplasmic RNA did not effectively compete for DNA binding sites, while nuclear RNA did. The results indicate a high degree of complexity for nuclear envelope-associated RNA, and are compatible with the idea that hnRNA may be processed after attachment to the nuclear envelope (or nuclear matrix).     

Ultrastructure of nuclear condensation and localization of DNA and proteins in spermatozoa of a dasyurid marsupial,Sminthopsis crassicaudata

In the dasyurid marsupial, Sminthopsis crassicaudata, the mature spermatozoon has an inner homogeneous (C1) and a peripheral indented (C2) region. Using DNase-gold conjugates, and biotinylated genomic DNA probes, DNA was found to occur in both C1 and C2 regions. The morphogenesis of the spermatozoon nucleus was investigated using ultrastructural and cytochemical studies. Spermiogenesis was divided into 15 steps. By step 10, condensation of the C1 region was complete, and at the caudal extremity of the spermatid nucleus, the nuclear envelope enclosed an electron-lucent space. This space and the surrounding nuclear envelope became very enlarged at step 11. At this stage, a plate of approximately 70 nm in thickness was present along the caudal segment of the C1 region; this “nuclear mantle” did not bind DNase-gold conjugates but stained for lysine-rich proteins using alcoholic phosphotungstic acid. Chromatin condensation resumed at step 12 with the appearance of spherical chromatin structures peripheral to the C1 chromatin. These structures then partially coalesced and the indentations of the C2 region were observed. The expanded nuclear envelope at the caudal extremity persisted in caput epididymal spermatozoa. Spherical inclusions within it did not bind to DNase-gold conjugates but stained for lysine-rich proteins. As the sperm traveled down the epididymis, these inclusions amassed near the nuclear pores and were then removed from the nucleus. In addition, the nuclear mantle was found to have disappeared by the time the spermatozoa reached the corpus epididymidis. © 1996 Wiley-Liss, Inc.     

Unusual nucleotide sequence of a DNA fragment isolated from nuclear envelopes of mouse hepatocytes

Fragments of chromosomal DNA isolated from nuclear envelopes of mouse hepatocytes were previously cloned and partially characterized in our laboratory. One of the cloned fragments (EnvM4) had unusual characteristics: abundant representability in the clone library (about 50%) and homology with DNA of archebacteria. An analysis of nucleotide sequence of this fragment conducted in more detail allowed the detection of an evolutionary conserved region present in the genomes of various organisms (from bacteria to human). Other characteristics of fragment EnvM4 suggest a possible functional role of chromosomal DNA regions determining the attachment of chromosomes to the nuclear envelope.     

Structure and expression of the maize (<Emphasis Type="Italic">Zea mays</Emphasis>L.) SUN-domain protein gene family: evidence for the existence of two divergent classes of SUN proteins in plants

Background  

The nuclear envelope that separates the contents of the nucleus from the cytoplasm provides a surface for chromatin attachment and organization of the cortical nucleoplasm. Proteins associated with it have been well characterized in many eukaryotes but not in plants. SUN (Sad1p/Unc-84) domain proteins reside in the inner nuclear membrane and function with other proteins to form a physical link between the nucleoskeleton and the cytoskeleton. These bridges transfer forces across the nuclear envelope and are increasingly recognized to play roles in nuclear positioning, nuclear migration, cell cycle-dependent breakdown and reformation of the nuclear envelope, telomere-led nuclear reorganization during meiosis, and karyogamy.     

Lamin activity is essential for nuclear envelope assembly in a Drosophila embryo cell-free extract

The role of the Drosophila lamin protein in nuclear envelope assembly was studied using a Drosophila in vitro assembly system that reconstitutes nuclei from added sperm chromatin or naked DNA. Upon incubation of the embryonic assembly extract with anti-Drosophila lamin antibodies, the attachment of nuclear membrane vesicles to chromatin surface and nuclear envelope formation did not occur. Lamina assembly and nuclear membrane vesicles attachment to the chromatin were inhibited only when the activity of the 75-kD lamin isoform was inhibited in both soluble and membrane-vesicles fractions. Incubation of decondensed sperm chromatin with an extract that was depleted of nuclear membranes revealed the presence of lamin molecules on the chromatin periphery. In addition, high concentrations of bacterially expressed lamin molecules added to the extract, were able to associate with the chromatin periphery, and did not inhibit nuclear envelope assembly. After nuclear reconstitution, a fraction of the lamin pool was converted into the typical 74- and 76-kD isoforms. Together, these data strongly support an essential role of the lamina in nuclear envelope assembly.     

Tau‐induced nuclear envelope invagination causes a toxic accumulation of mRNA in Drosophila

The nucleus is a spherical dual‐membrane bound organelle that encapsulates genomic DNA. In eukaryotes, messenger RNAs (mRNA) are transcribed in the nucleus and transported through nuclear pores into the cytoplasm for translation into protein. In certain cell types and pathological conditions, nuclei harbor tubular invaginations of the nuclear envelope known as the “nucleoplasmic reticulum.” Nucleoplasmic reticulum expansion has recently been established as a mediator of neurodegeneration in tauopathies, including Alzheimer's disease. While the presence of pore‐lined, cytoplasm‐filled, nuclear envelope invaginations has been proposed to facilitate the rapid export of RNAs from the nucleus to the cytoplasm, the functional significance of nuclear envelope invaginations in regard to RNA export in any disorder is currently unknown . Here, we report that polyadenylated RNAs accumulate within and adjacent to tau‐induced nuclear envelope invaginations in a Drosophila model of tauopathy. Genetic or pharmacologic inhibition of RNA export machinery reduces accumulation of polyadenylated RNA within and adjacent to nuclear envelope invaginations and reduces tau‐induced neuronal death. These data are the first to point toward a possible role for RNA export through nuclear envelope invaginations in the pathogenesis of a neurodegenerative disorder and suggest that nucleocytoplasmic transport machinery may serve as a possible novel class of therapeutic targets for the treatment of tauopathies.     

MAF1, a novel plant protein interacting with matrix attachment region binding protein MFP1, is located at the nuclear envelope.

The interaction of chromatin with the nuclear matrix via matrix attachment region (MAR) DNA is considered to be of fundamental importance for chromatin organization in all eukaryotic cells. MAR binding filament-like protein 1 (MFP1) from tomato is a novel plant protein that specifically binds to MAR DNA. Its filament protein-like structure makes it a likely candidate for a structural component of the nuclear matrix. MFP1 is located at nuclear matrix-associated, specklelike structures at the nuclear envelope. Here, we report the identification of a novel protein that specifically interacts with MFP1 in yeast two-hybrid and in vitro binding assays. MFP1 associated factor 1 (MAF1) is a small, soluble, serine/threonine-rich protein that is ubiquitously expressed and has no similarity to known proteins. MAF1, like MFP1, is located at the nuclear periphery and is a component of the nuclear matrix. These data suggest that MFP1 and MAF1 are in vivo interaction partners and that both proteins are components of a nuclear substructure, previously undescribed in plants, that connects the nuclear envelope and the internal nuclear matrix.     

The proliferating cell marker monoclonal antibody Ki-67 recognizes specific antigens associated with the nuclear envelope of the early Drosophila embryo

Summary— Immunofluorescence and immunoelectron microscopy indicated that the antibody raised against the nuclear antigen Ki-67 of mammalian cells recognized antigenic determinants of early Drosophila embryos, localized on the outside of the nuclear envelope. Hence, the nuclear envelope of Drosophila appears to share a similar epitope with the chromosome scaffold of mitotic mammalian cells. With the progression of mitosis the antigen persisted around the mitotic spindle region and was also found in the pole regions at metaphase and anaphase. The antibody also stained the equatorial regions of the spindles from anaphase to late telophase. The antibody may therefore be used as a biochemical marker of the nuclear envelope for studying nuclear membrane biogenesis and behavior during the mitotic divisions of the Drosophila embryo.     

Attachment of DNA Loops to an Artificial Matrix Does Not Affect the Replication Origin Specificity in Early Development of Xenopus laevis

Replication initiation proceeds in a random fashion in early development of Xenopus laevis. The replication origins become fixed only at later stages of development after the mid-blastula transition. Specification of replication origins occurs at the same time with the specification of the DNA attachment to the nuclear matrix. Replication origins of many species coincide or are located in the vicinity of sites of DNA attachment to the nuclear matrix. The present work was dedicated to development of an experimental system where DNA loops were specifically attached to an artificial matrix and a study of an effect of this attachment on specificity of DNA replication initiation in extracts of Xenopus laevis oocytes. We have found that DNA attachment to the artificial matrix increases the efficacy of DNA replication as compared to the control, but does not affect the replication specificity. It is likely that the transition from non-specific to specific replication is determined by a combination of several factors, and specificity of DNA attachment to a matrix alone is not sufficient for specification of a replication origin.     

COMPARATIVE CYTOLOGY OF ULOTHRIX AND STIGEOCLONIUM1

This investigation describes the cytology of the ulotrichalean genera Ulothrix and Stigeoclonium. Cellular organization is similar to the degree that interphase cells of the 2 genera cannot be distinguished with certainly. In Stigeoclonium, the nuclear envelope becomes disrupted at the end of prophase, and centrioles enter the nucleoplasm. At metaphase the nuclear envelope is again intact, and some of the spindle tubules appear to be contiguous with the nuclear envelope. The spindle in Ulothrix is essentially open with, no attachment of spindle tubules to the nuclear envelope and with, centrioles on the spindle-cytoplasm interface at the spindle poles. Spindle poles are blunt in Stigeoclonium and pointed in Ulothrix. Cytokinesis is by cell plate formation in both genera, but there is no phragmoplast.     

The role of the nuclear pore complex in adenovirus DNA entry.

Adenovirus targets its genome to the cell nucleus by a multistep process involving endocytosis, membrane penetration and cytoplasmic transport, and finally imports its DNA into the nucleus. Using an immunochemical and biochemical approach combined with inhibitors of nuclear import, we demonstrate that incoming viral DNA and DNA-associated protein VII enter the nucleus via nuclear pore complexes (NPCs). Depletion of calcium from nuclear envelope and endoplasmic reticulum cisternae by ionophores or thapsigargin blocked DNA and protein VII import into the nucleus, but had no effect on virus targeting to NPCs. Calcium-depleted cells were capable of disassembling incoming virus. In contrast, inhibitors of cytosolic O-linked glycoproteins of the NPC blocked virus attachment to the nuclear envelope, capsid disassembly and also nuclear import of protein VII. The data indicate that NPCs have multiple roles in adenovirus entry into cells: they contain a virus-binding and/or dissociation activity and provide a gateway for the incoming DNA genome into the nucleus.