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| 6. |
- Percipalle, P, et al.
(författare)
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Actin bound to the heterogeneous nuclear ribonucleoprotein hrp36 is associated with Balbiani ring mRNA from the gene to polysomes
- 2001
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Ingår i: The Journal of cell biology. - : Rockefeller University Press. - 0021-9525 .- 1540-8140. ; 153:1, s. 229-235
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Tidskriftsartikel (refereegranskat)abstract
- In the salivary glands of the dipteran Chironomus tentans, a specific messenger ribonucleoprotein (mRNP) particle, the Balbiani ring (BR) granule, can be visualized during its assembly on the gene and during its nucleocytoplasmic transport. We now show with immunoelectron microscopy that actin becomes associated with the BR particle concomitantly with transcription and is present in the particle in the nucleoplasm. DNase I affinity chromatography experiments with extracts from tissue culture cells indicate that both nuclear and cytoplasmic actin are bound to the heterogeneous RNP (hnRNP) protein hrp36, but not to the hnRNP proteins hrp23 and hrp45. The interaction is likely to be direct as purified actin binds to recombinant hrp36 in vitro. Furthermore, it is demonstrated by cross linking that nuclear as well as cytoplasmic actin are bound to hrp36 in vivo. It is known that hrp36 is added cotranscriptionally along the BR mRNA molecule and accompanies the RNA through the nuclear pores and into polysomes. We conclude that actin is likely to be bound to the BR transcript via hrp36 during the transfer of the mRNA from the gene all the way into polysomes.
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| 10. |
- Soop, T, et al.
(författare)
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A p50-like Y-box protein with a putative translational role becomes associated with pre-mRNA concomitant with transcription
- 2003
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Ingår i: Journal of Cell Science. - : The Company of Biologists. - 0021-9533 .- 1477-9137. ; 116:8, s. 1493-1503
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Tidskriftsartikel (refereegranskat)abstract
- In vertebrates free messenger ribonucleoprotein (RNP) particles and polysomes contain an abundant Y-box protein called p50 (YB-1), which regulates translation, presumably by affecting the packaging of the RNA. Here, we have identified a p50-like protein in the dipteran Chironomus tentans and studied its relation with the biogenesis of mRNA in larval salivary glands. The salivary gland cells contain polytene chromosomes with the transcriptionally active regions blown up as puffs. A few giant puffs, called Balbiani rings (BRs), generate a transcription product, a large RNP particle, which can be visualised (with the electron microscope) during its assembly on the gene and during its transport to and through the nuclear pores. The p50-like protein studied, designated Ct-p40/50 (or p40/50 for short), was shown to contain a central cold-shock domain, an alanine- and proline-rich N-terminal domain, and a C-terminal domain with alternating acidic and basic regions, an organisation that is characteristic of p50 (YB-1). The p40/50 protein appears in two isoforms, p40 and p50, which contain 264 and 317 amino acids, respectively. The two isoforms share the first 258 amino acids and thus differ in amino-acid sequence only in the region close to the C-terminus. When a polyclonal antibody was raised against p40/50, western blot analysis and immunocytology showed that p40/50 is not only abundant in the cytoplasm but is also present in the nucleus. Immunolabelling of isolated polytene chromosomes showed that p40/50 appears in transcriptionally active regions, including the BRs. Using immunoelectron microscopy we revealed that p40/50 is added along the nascent transcripts and is also present in the released BR RNP particles in the nucleoplasm. Finally, by UV crosslinking in vivo we showed that p40/50 is bound to both nuclear and cytoplasmic poly(A) RNA. We conclude that p40/50 is being added cotranscriptionally along the growing BR pre-mRNA, is released with the processed mRNA into the nucleoplasm and probably remains associated with the mRNA both during nucleocytoplasmic transport and protein synthesis. Given that the p40/p50 protein, presumably with a role in translation, is loaded onto the primary transcript concomitant with transcription, an early programming of the cytoplasmic fate of mRNA is indicated.
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| 13. |
- Yuan, L, et al.
(författare)
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The synaptonemal complex protein SCP3 can form multistranded, cross-striated fibers in vivo
- 1998
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Ingår i: The Journal of cell biology. - : Rockefeller University Press. - 0021-9525 .- 1540-8140. ; 142:2, s. 331-339
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Tidskriftsartikel (refereegranskat)abstract
- The synaptonemal complex protein SCP3 is part of the lateral element of the synaptonemal complex, a meiosis-specific protein structure essential for synapsis of homologous chromosomes. We have investigated the fiber-forming properties of SCP3 to elucidate its role in the synaptonemal complex. By synthesis of SCP3 in cultured somatic cells, it has been shown that SCP3 can self-assemble into thick fibers and that this process requires the COOH-terminal coiled coil domain of SCP3, as well as the NH2-terminal nonhelical domain. We have further analyzed the thick SCP3 fibers by transmission electron microscopy and immunoelectron microscopy. We found that the fibers display a transversal striation with a periodicity of ∼20 nm and consist of a large number of closely associated, thin fibers, 5–10 nm in diameter. These features suggest that the SCP3 fibers are structurally related to intermediate filaments. It is known that in some species the lateral elements of the synaptonemal complex show a highly ordered striated structure resembling that of the SCP3 fibers. We propose that SCP3 fibers constitute the core of the lateral elements of the synaptonemal complex and function as a molecular framework to which other proteins attach, regulating DNA binding to the chromatid axis, sister chromatid cohesion, synapsis, and recombination.
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| 15. |
- AlzhanovaEricsson, AT, et al.
(författare)
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A protein of the SR family of splicing factors binds extensively to exonic Balbiani ring pre-mRNA and accompanies the RNA from the gene to the nuclear pore
- 1996
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Ingår i: Genes & development. - : Cold Spring Harbor Laboratory. - 0890-9369 .- 1549-5477. ; 10:22, s. 2881-2893
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Tidskriftsartikel (refereegranskat)abstract
- We report on the molecular cloning and intracellular localization of a heterogeneous nuclear ribonucleoprotein (hnRNP), Ct-hrp45, one of the major components of pre-mRNP particles in Chironomus tentans. It is shown that hrp45 belongs to the SR family of splicing factors and exhibits high sequence similarity to Drosophila SRp55/B52 and human SF2/ASF. The distribution of hrp45 within the C. tentans salivary gland cells is studied by immunocytology. The hrp45 protein is found to be abundant in the nucleus, whereas it is undetectable in the cytoplasm. The fate of hrp45 in specific pre-mRNP particles, the Balbiani ring (BR) granules, is revealed by immunoelectron microscopy. It is observed that hrp45 is associated with the growing BR pre-mRNP particles and is being added continuously concomitant with the growth of the transcript, indicating that hrp45 is bound extensively to exon 4, which comprises 80-90% of the primary transcript. Furthermore, hrp45 remains bound to the BR RNP particles in the nucleoplasm and is not released until the particles translocate through the nuclear pore. Thus, hrp45 behaves as an hnRNP protein linked to exon RNA (and perhaps also to the introns) rather than as a spliceosome component connected to the assembly and disassembly of spliceosomes. It seems that hrp45, and possibly also other SR family proteins, is playing an important role in the structural organization of pre-mRNP particles and is perhaps participating not only in splicing but also in other intranuclear events.
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| 16. |
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| 17. |
- Daneholt, B
(författare)
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Packing and delivery of a genetic message
- 2001
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Ingår i: Chromosoma. - : Springer Science and Business Media LLC. - 0009-5915. ; 110:3, s. 173-185
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Tidskriftsartikel (refereegranskat)
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| 18. |
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| 19. |
- Hernandez-Hernandez, A, et al.
(författare)
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The central element of the synaptonemal complex in mice is organized as a bilayered junction structure
- 2016
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Ingår i: Journal of cell science. - : The Company of Biologists. - 1477-9137 .- 0021-9533. ; 129:11, s. 2239-2249
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Tidskriftsartikel (refereegranskat)abstract
- The synaptonemal complex (SC) transiently stabilizes pairing interactions between homologous chromosomes during meiosis. Assembly of the SC is mediated through integration of opposing transverse filaments (TF) into a central element (CE), a process that is poorly understood. We have here analyzed the localization of the TF protein SYCP1 and the CE proteins SYCE1, SYCE2 and SYCE3 within the central region of the SC in mouse spermatocytes using immunoelectron microscopy. Distribution of immuno-gold particles in a lateral view of the SC, supported by protein interaction data, suggest that the N-terminal region of SYCP1 and SYCE3 form a joint bilayered central structure and that SYCE1 and SYCE2 localize in between the two layers. We find that disruption of SYCE2 and TEX12 (a fourth CE protein) localization to the CE abolishes central alignment of the N-terminal region of SYCP1. Thus, our results show that all four CE proteins in an interdependent manner contribute to stabilization of opposing N-terminal regions of SYCP1, forming a bilayered TF-CE junction structure that promotes SC formation and synapsis.
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| 24. |
- MEHLIN, H, et al.
(författare)
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Structural interaction between the nuclear pore complex and a specific translocating RNP particle
- 1995
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Ingår i: The Journal of cell biology. - : Rockefeller University Press. - 0021-9525 .- 1540-8140. ; 129:5, s. 1205-1216
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Tidskriftsartikel (refereegranskat)abstract
- The transport of Balbiani ring (BR) premessenger RNP particles in the larval salivary gland cells of the dipteran Chironomus tentans can be followed using electron microscopy. A BR RNP particle consists of an RNP ribbon bent into a ringlike structure. Upon translocation through the nuclear pore complex (NPC), the ribbon is straightened and enters the central channel of the NPC with the 5' end of the transcript in the lead. The translocating ribbon is likely to interact with the central channel but, in addition, the remaining portion of the ribbon ring makes contact with the periphery of the NPC. To determine the nature of this latter interaction, we have now studied the connections between the RNP particle and the border of the NPC during different stages of translocation using electron microscope tomography. It was observed that the 3' terminal domain of the ribbon always touches the nuclear ring of the NPC, but the precise area of contact is variable. Sometimes also a region on the opposite side of the ribbon ring reaches the nuclear ring. The pattern of contacts could be correlated to the stage of translocation, and it was concluded that the particle-nuclear ring interactions reflect a rotation of the ribbon ring in front of the central channel, the rotation being secondary to the successive translocation of the ribbon through the channel. The particle's mode of interaction with the NPC suggests that the initial contact between the 5' end domain of the ribbon and the entrance to the central channel is probably crucial to accomplish the ordered translocation of the premessenger RNP particle through the NPC.
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| 25. |
- Narangifard, A., et al.
(författare)
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Human skin barrier formation takes place via a cubic to lamellar lipid phase transition as analyzed by cryo-electron microscopy and EM-simulation
- 2018
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Ingår i: Experimental Cell Research. - : Elsevier BV. - 0014-4827 .- 1090-2422. ; 366:2, s. 139-151
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Tidskriftsartikel (refereegranskat)abstract
- The skin's permeability barrier consists of stacked lipid sheets of splayed ceramides, cholesterol and free fatty acids, positioned intercellularly in the stratum corneum. We report here on the early stage of skin barrier formation taking place inside the tubuloreticular system in the secretory cells of the topmost viable epidermis and in the intercellular space between viable epidermis and stratum corneum. The barrier formation process was analysed in situ in its near-native state, using cryo-EM combined with molecular dynamics modeling and EM simulation. Stacks of lamellae appear towards the periphery of the tubuloreticular system and they are closely associated with granular regions. Only models based on a bicontinuous cubic phase organization proved compatible with the granular cryo-EM patterns. Only models based on a dehydrated lamellar phase organization agreed with the lamellar cryo-EM patterns. The data support that human skin barrier formation takes place via a cubic to lamellar lipid phase transition.
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| 30. |
- Rullgard, H., et al.
(författare)
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Simulation of transmission electron microscope images of biological specimens
- 2011
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Ingår i: Journal of Microscopy. - : Wiley. - 0022-2720 .- 1365-2818. ; 243:3, s. 234-256
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Tidskriftsartikel (refereegranskat)abstract
- We present a new approach to simulate electron cryo-microscope images of biological specimens. The framework for simulation consists of two parts; the first is a phantom generator that generates a model of a specimen suitable for simulation, the second is a transmission electron microscope simulator. The phantom generator calculates the scattering potential of an atomic structure in aqueous buffer and allows the user to define the distribution of molecules in the simulated image. The simulator includes a well defined electron-specimen interaction model based on the scalar Schrodinger equation, the contrast transfer function for optics, and a noise model that includes shot noise as well as detector noise including detector blurring. To enable optimal performance, the simulation framework also includes a calibration protocol for setting simulation parameters. To test the accuracy of the new framework for simulation, we compare simulated images to experimental images recorded of the Tobacco Mosaic Virus (TMV) in vitreous ice. The simulated and experimental images show good agreement with respect to contrast variations depending on dose and defocus. Furthermore, random fluctuations present in experimental and simulated images exhibit similar statistical properties. The simulator has been designed to provide a platform for development of new instrumentation and image processing procedures in single particle electron microscopy, two-dimensional crystallography and electron tomography with well documented protocols and an open source code into which new improvements and extensions are easily incorporated.
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| 35. |
- STARBORG, M, et al.
(författare)
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A murine replication protein accumulates temporarily in the heterochromatic regions of nuclei prior to initiation of DNA replication
- 1995
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Ingår i: Journal of cell science. - : The Company of Biologists. - 0021-9533 .- 1477-9137. ; 108108 ( Pt 3), s. 927-934
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Tidskriftsartikel (refereegranskat)abstract
- We have analyzed the expression of the murine P1 gene, the mammalian homologue of the yeast MCM3 protein, during the mitotic cell cycle. The MCM3 protein has previously been shown to be of importance for initiation of DNA replication in Saccharomyces cerevisiae. We found that the murine P1 protein was present in the nuclei of mammalian cells throughout interphase of the cell cycle. This is in contrast to the MCM3 protein, which is located in the nuclei of yeast cells only between the M and the S phase of the cell cycle. Detailed analysis of the intranuclear localization of the P1 protein during the cell cycle revealed that it accumulates transiently in the heterochromatic regions towards the end of G1. The accumulation of the P1 protein in the heterochromatic regions prior to activation of DNA replication suggests that the mammalian P1 protein is also of importance for initiation of DNA replication. The MCM2-3.5 proteins have been suggested to represent yeast equivalents of a hypothetical replication licensing factor initially described in Xenopus. Our data support this model and indicate that the murine P1 protein could function as replication licensing factor. The chromosomal localization of the P1 gene was determined by fluorescence in situ hybridization to region 6p12 in human metaphase chromosomes.
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| 36. |
- Sun, X, et al.
(författare)
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Conspicuous accumulation of transcription elongation repressor hrp130/CA150 on the intron-rich Balbiani ring 3 gene
- 2004
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Ingår i: Chromosoma. - : Springer Science and Business Media LLC. - 0009-5915 .- 1432-0886. ; 113:5, s. 244-257
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Tidskriftsartikel (refereegranskat)abstract
- Chromosomal puffs on the polytene chromosomes in the dipteran Chironomus tentans offer the possibility of comparing the appearance of RNA-binding proteins at different transcription sites. We raised a monoclonal antibody that recognized a 130 kDa protein, designated hrp130. Immunocytological analysis of isolated chromosomes showed that hrp130 is heavily accumulated in a specific puff, called Balbiani ring 3; only occasionally is hrp130 abundant in one or two additional puffs on other chromosomes. The immunolabeling was sensitive to RNase treatment, suggesting that hrp130 is associated with nascent ribonucleoproteins. As shown by immunoelectron microscopy hrp130 is distributed along the active BR3 genes. The full sequence of hrp130 was determined by cDNA cloning. The protein comprises 1028 amino acids and contains three WW domains in the N-terminal half and six FF domains in the C-terminal half of the molecule. The protein is conserved from Caenorhabditis elegans to mammals; the human homolog is known as the transcription elongation repressor CA150. We propose that the abundance of hrp130/CA150 in BR3 is connected with the exceptionally high level of splicing in this locus and that hrp130/CA150 adjusts the transcription rate to the numerous splicing events taking place along the gene to ensure proper splicing.
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| 37. |
- Sun, X, et al.
(författare)
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The hrp23 protein in the balbiani ring pre-mRNP particles is released just before or at the binding of the particles to the nuclear pore complex
- 1998
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Ingår i: The Journal of cell biology. - : Rockefeller University Press. - 0021-9525 .- 1540-8140. ; 142:5, s. 1181-1193
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Tidskriftsartikel (refereegranskat)abstract
- Balbiani ring (BR) pre-mRNP particles reside in the nuclei of salivary glands of the dipteran Chironomus tentans and carry the message for giant-sized salivary proteins. In the present study, we identify and characterize a new protein component in the BR ribonucleoprotein (RNP) particles, designated hrp23. The protein with a molecular mass of 20 kD has a single RNA-binding domain and a glycine-arginine-serine–rich auxiliary domain. As shown by immunoelectron microscopy, the hrp23 protein is added to the BR transcript concomitant with transcription, is still present in the BR particles in the nucleoplasm, but is absent from the BR particles that are bound to the nuclear pore complex or are translocating through the central channel of the complex. Thus, hrp23 is released just before or at the binding of the particles to the nuclear pore complex. It is noted that hrp23 behaves differently from two other BR RNP proteins earlier studied: hrp36 and hrp45. These proteins both reach the nuclear pore complex, and hrp36 even accompanies the RNA into the cytoplasm. It is concluded that each BR RNA-binding protein seems to have a specific flow pattern, probably related to the particular role of the protein in gene expression.
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| 38. |
- Thiry, M, et al.
(författare)
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Evaluation of the sensitivity of the terminal deoxynucleotidyl transferase-immunogold technique on Balbani ring genes
- 1998
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Ingår i: The journal of histochemistry and cytochemistry : official journal of the Histochemistry Society. - : SAGE Publications. - 0022-1554. ; 46:3, s. 345-351
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Tidskriftsartikel (refereegranskat)abstract
- Recently, we developed the terminal deoxynucleotidyl transferase (TdT)–immunogold technique for in situ detection of DNA molecules. In this study the potential value and the limitations of the method were evaluated using the giant polytene chromosomes from Chironomus tentans salivary glands. Emphasis was put on the Balbiani rings (BRs), specialized chromosomal sites with exceptionally intense synthesis of large mRNA molecules. Immunolabeling was recorded not only over the bands and interbands of the polytene chromosomes but also over the BR structures. In the BRs, gold particles were present over segments of active transcription units, each with a central chromatin axis and a number of growing RNP products attached to the axis. One third of the transversely sectioned transcription units showed labeling in the central parts, i.e., where the unfolded chromatin axis is located, whereas the growing RNP fibers remained unlabeled. The absence of labeling of the RNP fibers is not likely to be due to lack of accessibility, because anti-RNA antibodies readily decorated the RNP fibers. The nuclear sap and cytoplasm displayed no significant label. These results clearly indicate that the TdT–immunogold technique is specific for DNA and detects not only DNA in compacted chromatin but also fully extended DNA. Its ability to efficiently label a single DNA molecule demonstrates the method's very high sensitivity.
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| 39. |
- Visa, N, et al.
(författare)
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A nuclear cap-binding complex binds Balbiani ring pre-mRNA cotranscriptionally and accompanies the ribonucleoprotein particle during nuclear export
- 1996
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Ingår i: The Journal of cell biology. - : Rockefeller University Press. - 0021-9525 .- 1540-8140. ; 133:1, s. 5-14
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Tidskriftsartikel (refereegranskat)abstract
- In vertebrates, a nuclear cap-binding complex (CBC) formed by two cap- binding proteins, CBP20 and CBP80, is involved in several steps of RNA metabolism, including pre-mRNA splicing and nuclear export of some RNA polymerase II-transcribed U snRNAs. The CBC is highly conserved, and antibodies against human CBP20 cross-react with the CBP20 counterpart in the dipteran Chironomus tentans. Using immunoelectron microscopy, the in situ association of CBP20 with a specific pre-mRNP particle, the Balbiani ring particle, has been analyzed at different stages of pre-mRNA synthesis, maturation, and nucleo-cytoplasmic transport. We demonstrate that CBP20 binds to the nascent pre-mRNA shortly after transcription initiation, stays in the RNP particles after splicing has been completed, and remains attached to the 5' domain during translocation of the RNP through the nuclear pore complex (NPC). The rapid association of CBP20 with nascent RNA transcripts in situ is consistent with the role of CBC in splicing, and the retention of CBC on the RNP during translocation through the NPC supports its proposed involvement in RNA export.
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