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Re-localization of nuclear DNA helicase II during the growth period of bovine oocytes 总被引:2,自引:2,他引:0
Nuclear DNA helicase II (NDH II) is the bovine homolog of human RNA helicase A. The aim of this study was to compare NDH II
localization between somatic cells (bovine embryonal fibroblasts) and female germ cells (oocytes), with the main focus on
the dynamic changes in the redistribution of NDH II during the growth phase of the bovine oocytes. The fine granular staining
of NDH II was spread in the whole nucleoplasm of fibroblasts, excluding the reticulated nucleoli. In contrast, the large reticulated
nucleoli of the growing oocytes isolated from early antral follicles exhibited strong positivity for NDH II together with
the immunostaining signals of upstream binding factor (UBF) and RNA polymerase I subunit (PAF53), documenting the high synthetic
activity of these nucleoli. At the time of termination of oocyte growth, NDH II was preferentially located at the nucleolar
periphery together with proteins of fibrillar centres. In fully grown oocytes, NDH II was still present in the thin periphery
shell around the compact nucleolar core. The semiquantitative RT-PCR revealed that the average signal of NDH II mRNA in fully
grown oocytes was only at 40% level in comparison with growing oocytes. Western blot analysis further confirmed that a 140 kD
NDH II protein was abundant in growing oocytes, while the signal was substantially weaker in fully grown oocytes. The significant
decrease in NDH II gene expression and in NDH II mRNA translation correlates with a termination of the oocyte growth. Altogether,
the results demonstrate that NDH II expression parallels the activity of ribosomal RNA biosynthesis in the bovine growing
oocytes. 相似文献
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The genes of rRNA in the nucleolar organizer region (NOR) are inactivated in the oocytes of adult birds despite the functioning of lampbrush chromosomes. The nucleolus is not formed during all stages of the oocyte development. On the other hand, two morphological forms of oocytes differing by the presence of nucleolus in the germinal vesicle are described in the ovaries of juvenile birds. The activation and function of the ribosomal genes in avian oogenesis is still vague. In this work, the NOR activation in chicken (Gallus gallus domesticus) oocytes is confirmed with the help of fluorescence immunohistochemistry (antibodies against nucleophosmin, fibrillarin, and UBF1) and in situ nucleic acid hybridization (FISH with the probe to ITS1 in pre-rRNA). It is demonstrated that the nucleolus in the oocytes at the lampbrush stage in the chicken ovaries is fragmented after complete inactivation of the ribosome genes: the nucleolar fragments contain fibrillarin but do not contain pre-rRNA molecule. The utility of the ovary 3D reconstruction using serial histological sections for quantification of sex cell population heterogeneity in the ovaries of juvenile birds is demonstrated. The obtained results improve the current insight into the functional NOR state in the oocytes of juvenile female birds and contribute to the concept of diversity in the scenarios of gametogenesis. 相似文献
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The Nopp140 gene of Drosophila maps within 79A5 of chromosome 3. Alternative splicing yields two variants. DmNopp140 (654 residues) is the sequence homolog of vertebrate Nopp140. Its carboxy terminus is 64% identical to that of the prototypical rat Nopp140. DmNopp140-RGG (688 residues) is identical to DmNopp140 throughout its first 551 residues, but its carboxy terminus contains a glycine/arginine-rich domain that is often found in RNA-binding proteins such as vertebrate nucleolin. Both Drosophila variants localize to nucleoli in Drosophila Schneider II cells and Xenopus oocytes, specifically within the dense fibrillar components. In HeLa cells, DmNopp140-RGG localizes to intact nucleoli, whereas DmNopp140 partitions HeLa nucleoli into phase-light and phase-dark regions. The phase-light regions contain DmNopp140 and endogenous fibrillarin, whereas the phase-dark regions contain endogenous nucleolin. When coexpressed, both Drosophila variants colocalize to HeLa cell nucleoli. Both variants fail to localize to endogenous Cajal bodies in Xenopus oocyte nuclei and in HeLa cell nuclei. Endogenous HeLa coilin, however, accumulates around the periphery of phase-light regions in cells expressing DmNopp140. The carboxy truncation (DmNopp140DeltaRGG) also fails to localize to Cajal bodies, but it forms similar phase-light regions that peripherally accumulate endogenous coilin. Conversely, we see no unusual accumulation of coilin in cells expressing DmNopp140-RGG. 相似文献
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We expressed two green fluorescent protein (GFP)-tagged Nopp140 isoforms in transgenic Drosophila melanogaster to study nucleolar dynamics during oogenesis and early embryogenesis. Specifically, we wanted to test whether the quiescent
oocyte nucleus stored maternal Nopp140 and then to determine precisely when nucleoli formed during embryogenesis. During oogenesis
nurse cell nucleoli accumulated GFP-Nopp140 gradually such that posterior nurse cell nucleoli in egg chambers at stage 10
were usually brighter than the more anterior nurse cell nucleoli. Nucleoli within apoptotic nurse cells disassembled in stages
12 and 13, but not all GFP-Nopp140 entered the oocyte through inter-connecting cytoplasmic bridges. Oocytes, on the other
hand, lost their nucleoli by stage 3, but GFP-Nopp140 gradually accumulated in oocyte nuclei during stages 8–13. Most oocyte
nuclei at stage 10 stored GFP-Nopp140 uniformly, but many stage 10 oocytes accumulated GFP-Nopp140 in presumed endobodies
or in multiple smaller spheres. All oocyte nuclei at stages 11-12 were uniformly labeled, and GFP-Nopp140 diffused to the
cytoplasm upon nuclear disassembly in stage 13. GFP-Nopp140 reappeared during embryogenesis; initial nucleologenesis occurred
in peripheral somatic nuclei during embryonic stage 13, one stage earlier than reported previously. These GFP-Nopp140-containing
foci disassembled at the 13th syncytial mitosis, and a second nucleologenesis occurred in early stage 14. The resulting nucleoli
occupied nuclear regions closest to the periphery of the embryos. Pole cells contained GFP-Nopp140 during the syncytial embryonic
stages, but their nucleologenesis started at gastrulation.
This work was supported by the National Science Foundation (grant MCB-0234245). O'Keith Dellafosse was supported by the Louisiana
Alliance for Minority Participation (LAMP). 相似文献
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A class of nonribosomal nucleolar components is located in chromosome periphery and in nucleolus-derived foci during anaphase and telophase 总被引:9,自引:0,他引:9
Miroslav Dundr U. Thomas Meier Nancy Lewis David Rekosh Marie-Louise Hammarskjöld Mark O. J. Olson 《Chromosoma》1997,105(7-8):407-417
The subcellular location of several nonribosomal nucleolar proteins was examined at various stages of mitosis in synchronized
mammalian cell lines including HeLa, 3T3, COS-7 and HIV-1 Rev-expressing CMT3 cells. Nucleolar proteins B23, fibrillarin,
nucleolin and p52 as well as U3 snoRNA were located partially in the peripheral regions of chromosomes from prometaphase to
early telophase. However, these proteins were also found in large cytoplasmic particles, 1–2 μm in diameter, termed nucleolus-derived
foci (NDF). The NDF reached maximum numbers (as many as 100 per cell) during mid- to late anaphase, after which their number
declined to a few or none during late telophase. The decline in the number of NDF approximately coincided with the appearance
of prenucleolar bodies and reforming nucleoli. The HIV-1 Rev protein and a mutant Rev protein defective in its nuclear export
signal were also found in the NDF. The mutant Rev protein precisely followed the pattern of localization of the above nucleolar
proteins, whereas the wild-type Rev did not enter nuclei until G1 phase. The nucleolar shuttling phosphoprotein Nopp 140 did
not follow the above pattern of localization during mitosis: it dispersed in the cytoplasm from prometaphase through early
telophase and was not found in the NDF. Although the NDF and mitotic coiled bodies disappeared from the cytoplasm at approximately
the same time during mitosis, protein B23 was not found in mitotic coiled bodies, nor was p80 coilin present in the NDF. These
results suggest that a class of proteins involved in preribosomal RNA processing associate with chromosome periphery and with
NDF as part of a system to conserve and deliver preexisting components to reforming nucleoli during mitosis.
Edited by: S. A. Gerbi 相似文献
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Fibrillarin is one of the major nucleolar proteins and is involved in pre-rRNA maturation. Its three main regions are a glycine and arginine-rich (GAR) domain, an RNA-binding domain, and an alpha-helical region, which presumably has a methyltransferase activity. Yet the roles of these regions in nucleolus-specific localization of fibrillarin are still unclear. To elucidate this issue, a series of plasmids was constructed to express human fibrillarin mutants fused with the green fluorescent protein. Localization of the chimeric proteins was studied in interphase and mitotic HeLa cells after single transfection with the plasmids. Deletion or a mutation of any domain proved to alter the specific fibrillarin location coinciding with sites of pre-rRNA synthesis. The GAR domain and the first spacer together were sufficient for fibrillarin migration into the nucleolus. Fibrillarin mutants located within the interphase nucleolus did not differ in mitotic location from the wild-type fibrillarin. 相似文献
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Characterization and differential nuclear localization of Nopp140 and a novel Nopp140-like protein in trypanosomes
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Trypanosomatids possess two homologues of Nopp140: a canonical Nopp140 and a Nopp140-like protein (TbNoLP) in which a GAR domain replaces the C-terminal SRP40 domain. Both are phosphorylated and coimmunoprecipitate with RNA polymerase I. Each paralogue has a distinct subnuclear localization, and depletion of TbNoLP produces an enlarged nucleolus in which TbNopp140-containing regions disperse. The restricted occurrence pattern of NoLP proteins reflects an intriguing convergence in evolution, suggestive of a function in nucleoplasmic small nucleolar ribonucleoprotein shuttling. 相似文献
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Muro E Hoang TQ Jobart-Malfait A Hernandez-Verdun D 《Biology of the cell / under the auspices of the European Cell Biology Organization》2008,100(5):303-313
Background information. The nucleolus is a dynamic structure. It has been demonstrated that nucleolar proteins rapidly associate with and dissociate from nucleolar components in continuous exchanges with the nucleoplasm using GFP (green fluorescent protein)‐tagged proteins. However, how the exchanges within one nucleolus and between nucleoli within the nuclear volume occurred is still poorly understood. Results. The movement of PAGFP (photoactivatable GFP)‐tagged proteins that become visible after photoactivation can be followed. In the present study, we establish the protocol allowing quantification of the traffic of PAGFP‐tagged nucleolar proteins in nuclei containing two nucleoli. The traffic in the activated area, at the periphery of the activated area and to the neighbouring nucleolus is measured. Protein B23 is rapidly replaced in the activated area, and at the periphery of the activated area the steady state suggests intranucleolar recycling of B23; this recycling is LMB (leptomycin B)‐sensitive. The pool of activated B23 is equally distributed in the volume of the two nucleoli within 2 min. The three‐dimensional distribution of the proteins Nop52 and fibrillarin is less rapid than that of B23 but is also LMB‐sensitive. In contrast, traffic of fibrillarin from the nucleoli to the CB (Cajal body) was not modified by LMB. Conclusions. We propose that the steady state of nucleolar proteins in nucleoli depends on the affinity of the proteins for their partners and on intranucleolar recycling. This steady state can be impaired by LMB but not the uptake in the neighbouring nucleolus or the CB. 相似文献
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Eleonora Muro Thang Q Hoang Aude Jobart-Malfait Danièle Hernandez-Verdun 《Biology of the cell / under the auspices of the European Cell Biology Organization》2008,100(5):303-313
BACKGROUND INFORMATION: The nucleolus is a dynamic structure. It has been demonstrated that nucleolar proteins rapidly associate with and dissociate from nucleolar components in continuous exchanges with the nucleoplasm using GFP (green fluorescent protein)-tagged proteins. However, how the exchanges within one nucleolus and between nucleoli within the nuclear volume occurred is still poorly understood. RESULTS: The movement of PAGFP (photoactivatable GFP)-tagged proteins that become visible after photoactivation can be followed. In the present study, we establish the protocol allowing quantification of the traffic of PAGFP-tagged nucleolar proteins in nuclei containing two nucleoli. The traffic in the activated area, at the periphery of the activated area and to the neighbouring nucleolus is measured. Protein B23 is rapidly replaced in the activated area, and at the periphery of the activated area the steady state suggests intranucleolar recycling of B23; this recycling is LMB (leptomycin B)-sensitive. The pool of activated B23 is equally distributed in the volume of the two nucleoli within 2 min. The three-dimensional distribution of the proteins Nop52 and fibrillarin is less rapid than that of B23 but is also LMB-sensitive. In contrast, traffic of fibrillarin from the nucleoli to the CB (Cajal body) was not modified by LMB. CONCLUSIONS: We propose that the steady state of nucleolar proteins in nucleoli depends on the affinity of the proteins for their partners and on intranucleolar recycling. This steady state can be impaired by LMB but not the uptake in the neighbouring nucleolus or the CB. 相似文献
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Mutational analysis of p80 coilin indicates a functional interaction between coiled bodies and the nucleolus
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《The Journal of cell biology》1995,131(4):817-831
Coiled bodies are conserved subnuclear domains found in both plant and animal cells. They contain a subset of splicing snRNPs and several nucleolar antigens, including Nopp140 and fibrillarin. In addition, autoimmune patient sera have identified a coiled body specific protein, called p80 coilin. In this study we show that p80 coilin is ubiquitously expressed in human tissues. The full-length human p80 coilin protein correctly localizes in coiled bodies when exogenously expressed in HeLa cells using a transient transfection assay. Mutational analysis identifies separate domains in the p80 coilin protein that differentially affect its subnuclear localization. The data show that p80 coilin has a nuclear localization signal, but this is not sufficient to target the protein to coiled bodies. The results indicate that localization in coiled bodies is not determined by a simple motif analogous to the NLS motifs involved in nuclear import. A specific carboxy-terminal deletion in p80 coilin results in the formation of pseudo-coiled bodies that are unable to recruit splicing snRNPs. This causes a loss of endogenous coiled bodies. A separate class of mutant coilin proteins are shown to localize in fibrillar structures that surround nucleoli. These mutants also lead to loss of endogenous coiled bodies, produce a dramatic disruption of nucleolar architecture and cause a specific segregation of nucleolar antigens. The structural change in nucleoli is accompanied by the loss of RNA polymerase I activity. These data indicate that p80 coilin plays an important role in subnuclear organization and suggest that there may be a functional interaction between coiled bodies and nucleoli. 相似文献