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PubMed1997

Experimental induction of prenucleolar bodies (PNBs) in interphase cells: interphase PNBs show similar characteristics as those typically observed at telophase of mitosis in untreated cells.

Recently, it was shown that a short exposure of living mammalian cells to low ionic strength buffers (hypotonic shock) caused partial or almost complete unraveling of interphase nucleoli. However, when the cells were released from the hypotonic shock and transferred to normal isotonic medium, functionally active and structurally integral nucleoli were reassembled at their initial positions within interphase nuclei. Here, we show further that this process is accompanied by the appearance of numerous discrete extranucleolar bodies, which have striking similarities to the prenucleolar bodies (PNBs) observed in untreated cells at telophase of mitosis. (1) Like PNBs at mitosis, hypotonically induced interphase PNBs are composed of RNA-positive granules and fibrils, contain the major nucleolar protein B23 and silver-binding proteins, but lack DNA and RNA polymerase I transcription factor UBF. (2) As for mitotic PNBs, disappearance of the interphase PNB counterparts coincides with the increase in size of reconstructed nucleoli. (3) Addition of actinomycin D does not prevent assembly of interphase PNBs, but does arrest their coalescence with the chromosomal nucleolus-organizing regions and blocks the complete reformation of nucleoli. It is concluded that the assembly of PNBs generally observed at telophase of mitosis can be induced experimentally in nuclei of interphase mammalian cells in vivo. At interphase, this process is probably initiated by changes in the intracellular ionic environment.

Animals↗
PubMed1997

Relative distribution of rDNA and proteins of the RNA polymerase I transcription machinery at chromosomal NORs.

Using confocal and immunofluorescence microscopy the relative distribution of the ribosomal chromatin and some proteins of the RNA polymerase I transcription machinery such as upstream binding factor (UBF), RNA polymerase I and DNA topoisomerase I was analyzed on chromosomal nucleolus organizer regions (NORs) of PtK1 cells. Staining with various DNA fluorochromes revealed that the ribosomal chromatin may be found at the axial region of the NOR and also at lateral expansions around the axis that can also be detected by in situ hybridization. It was observed that the transcription machinery shows a crescent-shaped distribution around the axial ribosomal chromatin at the NOR of metaphase and anaphase chromatids. An ultrastructural analysis of serially sectioned NORs supports this crescent-shape organization. Taking into account previous and present results and the loop/scaffold model of chromosome structure, we propose a model of NOR organization. The model proposes that ribosomal genes that were inactive in the preceding interphase would be present as condensed short Q-loops occupying the axial region of the NOR. Ribosomal genes previously active during interphase would be undercondensed as large R-loops associated with the transcription machinery, which is distributed in a crescent-shaped fashion around the previously active ribosomal DNA.

Anaphase↗
PubMed1999

Diversification pattern of the HMG and SOX family members during evolution.

From a database containing the published HMG protein sequences, we constructed an alignment of the HMG box functional domain based on sequence identity. Due to the large number of sequences (more than 250) and the short size of this domain, several data sets were used. This analysis reveals that the HMG box superfamily can be separated into two clearly defined subfamilies: (i) the SOX/MATA/TCF family, which clusters proteins able to bind to specific DNA sequences; and (ii) the HMG/UBF family, which clusters members which bind non specifically to DNA. The appearance and diversification of these subfamilies largely predate the split between the yeast and the metazoan lineages. Particular emphasis was placed on the analysis of the SOX subfamily. For the first time our analysis clearly identified the SOX subfamily as structured in six groups of genes named SOX5/6, SRY, SOX2/3, SOX14, SOX4/22, and SOX9/18. The validity of these gene clusters is confirmed by their functional characteristics and their sequences outside the HMG box. In sharp contrast, there are only a few robust branching patterns inside the UBF/HMG family, probably because of the much more ancient diversification of this family than the diversification of the SOX family. The only consistent groups that can be detected by our analysis are HMG box 1, vertebrate HMG box 2, insect SSRP, and plant HMG. The various UBF boxes cannot be clustered together and their diversification appears to be extremely ancient, probably before the appearance of metazoans.

Animals↗
PubMed2005

Re-localization of nuclear DNA helicase II during the growth period of bovine oocytes.

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.

Adenosine Triphosphatases↗
PubMed2005

DADLE induces a reversible hibernation-like state in HeLa cells.

[D-Ala(2)-D-Leu(5)-Enkephalin] (DADLE) can induce hibernation when injected into ground squirrels in summer and is able to increase the survival time of explanted organs such as liver and lung. Since cell metabolism is a target of this peptide, we have treated HeLa cells with DADLE and investigated its possible effect on transcription and proliferation as well as the resumption of metabolic activity after treatment. The labelling for Pol I, Pol II and for splicing factors such as snRNPs and SC-35 decreased after treatment as did the nucleolar labelling for UBF. In treated cells, several spherical nuclear bodies were found to be labelled for hnRNPs. In parallel, the number of proliferating cells decreased after treatment with DADLE. After recovery, there was a gradual resumption of cell function: transcription and splicing factors had a distribution similar to that of controls; proliferation resumed; nuclear bodies, representing storage sites for RNPs, disappeared.

Antimetabolites↗
PubMed2000

Ultrastructural analysis of nucleolar transcription in cells microinjected with 5-bromo-UTP.

In situ sites of nucleolar transcription in cells microinjected with 5-bromo-UTP (BrUTP) were visualized at an ultrastructural level. After injection the cells were maintained for 4-90 min at 37 degrees C, fixed, and embedded in LR White resin. Postembedding immunoelectron microscopic visualization with colloidal gold has been used for localizing both Br-labeled precursor incorporated into pre-rRNA and different nucleolar transcription or processing factors. This high resolution approach allowed us to identify significant signal as early as after 4-min incubation periods following BrUTP microinjection. It revealed the dense fibrillar component (DFC) as being the first nucleolar compartment labeled with anti-bromodeoxyuridine antibody. Moreover, RNA polymerase I, nucleolar transcription factor UBF, and fibrillarin were also detected almost exclusively in this same nucleolar compartment. From 30 min onward, following microinjection, Br-labeled rRNA occurred also in the granular component. The results indicate that the DFC is the site of pre-rRNA transcription and of initial steps of pre-rRNA processing. Moreover, it demonstrates that BrUTP microinjection followed by postembedding detection of Br-labeled RNA is a useful technique for high resolution studies of structure-function associations in the nucleolus.

Cell Nucleolus↗
PubMed1980

Uterine and ovarian artery blood flow in the rhesus monkey near term.

Uterine and ovarian artery blood flow (UABF and OABF) were measured in six pregnant monkeys near term by means of the electromagnetic flowmeter. The average UABF was 21.4 ml/min and OABF was 1.9 ml/min. Complete aortic occlusion reduced UABF from 21.4 to 2.4 ml/min, while OABF increased from 1.9 to only 2.1 ml/min. In six pregnant monkeys near term, chronic partial constriction of the abdominal aorta for an average of 10 days produced a parallel reduction in UABF but no increased circulation through the ovarian or round ligament arteries. The ovarian arteries were not visualized on angiography in these pregnant monkeys even when the abdominal aorta was acutely or chronically constricted; the uterine arteries were readily seen. The authors concluded that acute or chronic aortic constriction in the pregnant monkey at term results in a noncompensated uterine blood flow (UBF) reduction. The possible implications in pregnant women are discussed.

Animals↗
PubMed1981

Uterine blood flow distribution after indomethacin infusion in the pregnant rabbit.

The distribution of uterine blood flow (UBF) in the chronically instrumented pregnant term rabbit was examined before and after indomethacin infusion. A significant fall in placental but not myometrial blood flow was observed. The fall in placental blood flow was significantly higher in placentas which were implanted in the midsection of the uterine horn than in those implanted in both ends of the horn. The results suggest that different physiologic mechanisms may regulate the blood flows in the placenta and the myometrium.

Animals↗
PubMed1993

Molecular characterization of the largest subunit of Plasmodium falciparum RNA polymerase I.

Plasmodium species possess developmentally regulated ribosomal RNA (rRNA) genes. This report describes the expression and gene structure of the largest subunit of P. falciparum RNA polymerase I (RNAPI), which is responsible for the synthesis of rRNA. The RNAPI largest subunit gene was present as a single copy gene on chromosome 9. Three exons encode the 2910-amino acid RNAPI polypeptide (340 140 Da). A comparison of Plasmodium, Trypanosoma, and Saccharomyces cerevisiae nuclear RNAP largest subunits identified conserved amino acid positions and class-specific amino acid positions. Novel amino acid insertions were found between RNAPI conserved regions A and B (region A'), D and DE1 (region D'), DE2 and E (region DE2'), and F and G (region F'). Leucine zipper domains were found within regions D', DE2, and DE2'. A novel serine-rich repeat domain, a domain with homology to the C-terminal domain of eukaryotic upstream binding factor (UBF), and 4 highly conserved casein kinase II (CKII) Ser/Thr phosphorylation motifs were found within a 127-amino acid sub-region of enlarged region F'. The novel RNAPI serine-rich repeat contained a conserved motif, Ser-X3-Ser, which was also identified in the serine-rich repeat domains of the P. falciparum RNAPII and RNAPIII largest subunits, as well as within a highly homologous serine-rich repeat from trophozoite antigen R45. The results of this molecular analysis indicate that phosphorylation and dephosphorylation mechanisms regulate the activity of P. falciparum RNAPI.

Amino Acid Sequence↗
PubMed1993

Characterization of the 130-bp repeat enhancer element of the rat ribosomal gene: functional interaction with transcription factor E1BF.

The 130-bp repetitive element (RE) of the rat rDNA (ribosomal RNA-encoding gene) intergenic spacer stimulated the synthesis of rRNA four- to sixfold, in comparison with that of the promoter alone, both in vivo and in vitro, when ligated to the rat rDNA promoter. The addition of increasing amounts of highly purified E1BF (enhancer-1 binding factor), which binds to the rat rDNA promoter and an upstream nonrepetitive enhancer element [Zhang and Jacob, Mol. Cell. Biol. 10 (1990) 5177-5186], to an in vitro transcription system resulted in enhancement of rDNA transcription from the recombinant plasmids containing the promoter or promoter-RE. However, E1BF-mediated stimulation of transcription under the influence of the RE continued at higher concentrations of E1BF than did the control transcription from the promoter alone. The binding affinity of E1BF for the RE was comparable to its affinity for the nonrepetitive far upstream enhancer element previously characterized in our laboratory. The sequences protected by E1BF in the RE differed from those protected by UBF (upstream control element-binding factor), a well characterized pol I transcription factor. These data suggest that E1BF belongs to a class of transcription factors which interact with the promoter and spacer cis-acting RE to modulate rDNA transcription.

Animals↗
PubMed2004

Regulation of ribosomal RNA gene expression in porcine oocytes.

In vitro production (IVP) of porcine embryos including in vitro maturation (IVM) of oocytes followed by in vitro fertilization (IVF) and in vitro culture (IVC) of the resultant embryos may result in live offspring, but it is still associated with great inefficiencies probably due to incomplete cytoplasmic maturation of the oocytes in vitro. Therefore, fundamental knowledge on the regulation of transcription during the oocyte growth phase when the messengers and protein synthetic machinery necessary for oocyte developmental competence are formed, is of great importance. In mammals, synthesis of RNA, up to 60-70% of which is ribosomal (rRNA), increases during oocyte growth and reaches a peak at the beginning of follicular antrum formation. In oocytes at the end of the growth phase, acquisition of full meiotic competence coincides with a markedly decreased rRNA transcriptional activity in the gametes. Our recent studies on the porcine oocyte growth phase have revealed a deeper molecular and biological insight into the complex regulation of rRNA transcription at different stages of follicular development. The data indicate that the so-called pocket protein, p130, is involved in the down-regulation of rRNA transcription at the end of the oocyte growth phase through an inhibition of the action of upstream binding factor (UBF). The latter protein is necessary for the function of RNA polymerase I (RNA Pol I), which is the actual enzyme driving rRNA gene transcription. Moreover, rRNA transcription also appears to be down-regulated by a decrease in the expression of mRNA encoding PAF53, an RNA Pol I-associated factor also required for the polymerase to exert its action. At the ultrastructural level, these molecular changes are paralleled by marginalization of the fibrillar centres of the oocyte nucleolus followed by compaction of the nucleolus into an inactive sphere of fibrils.

Animals↗
PubMed2004

Aging blunts remodeling of the uterine artery during murine pregnancy.

OBJECTIVE: The progressive increase in uterine blood flow (UBF) during pregnancy is accommodated by morphologic changes in the uterine artery (UA) in a process defined as arterial remodeling. This process is accompanied by changes in cytoskeletal architecture of the arterial smooth muscle cells (SMCs) and surrounding extracellular matrix (ECM). Aging reduces flow-induced arterial remodeling. We studied changes in the murine UA during pregnancy and on the effects of aging on the capacity of the UA to remodel in response to pregnancy. METHODS: We determined morphologic and cytologic changes in UA from nonpregnant and pregnant mice aged 12 weeks (young) and 40 weeks (old) and correlated them with their reproductive performance. RESULTS: In young mice, pregnancy induced an early increase in UA wall mass, which preceded lumen widening. These changes were not accompanied by altered densities of elastin and collagen in the ECM of the medial layer. Smooth muscle cell proliferation increased in midpregnancy and was paralleled by a transient decrease in smoothelin and smooth muscle alpha-actin expression. In old mice, these pregnancy-dependent changes in the UA wall were either absent or markedly reduced. Although by day 11 of pregnancy litter size did not differ between both age groups, the number of viable pups in old mice by day 17 of pregnancy and at birth was 25% and 60% less than in young mice. CONCLUSION: Outward hypertrophic remodeling of the UA during pregnancy in young mice is characterized by transient phenotypic modulation and proliferation of SMCs and alterations in the composition of the ECM. In contrast, in older mice, UA remodeling is markedly reduced and accompanied with a loss of viable fetuses near term pregnancy.

Aging↗
PubMed2004

Nuclear DNA helicase II (RNA helicase A) binds to an F-actin containing shell that surrounds the nucleolus.

Nuclear DNA helicase II (NDH II), alternatively named RNA helicase A (RHA), is an F-actin binding protein that is particularly enriched in the nucleolus of mouse cells. Here, we show that the nucleolar localization of NDH II of murine 3T3 cells depended on an ongoing rRNA synthesis. NDH II migrated out of the nucleolus after administration of 0.05 microg/ml actinomycin D, while nucleolin and the upstream binding factor (UBF) remained there. In S phase-arrested mouse cells, NDH II was frequently found at the nucleolar periphery, where it was accompanied by newly synthesized nucleolar RNA. Human NDH II was mainly distributed through the whole nucleoplasm and not enriched in the nucleoli. However, in the human breast carcinoma cell line MCF-7, NDH II was also found at the nucleolar periphery, together with the tumor suppressor protein p53. Both NDH II and p53 were apparently attached to the F-actin-based filamentous network that surrounded the nucleoli. Accordingly, this subnuclear structure was sensitive to F-actin depolymerizing agents. Depolymerization with gelsolin led to a striking accumulation of NDH II in the nucleoli of MCF-7 cells. This effect was abolished by RNase, which extensively released nucleolus-bound NDH II when added together with gelsolin. Taken together, these results support the idea that an actin-based filamentous network may anchor NDH II at the nucleolar periphery for pre-ribosomal RNA processing, ribosome assembly, and/or transport.

Actins↗
PubMed1998

Differential effects of heavy metals on the binding of Xenopus upstream binding factor(xUBF) to DNA.

Xenopus upstream binding factor (xUBF) is a transcription factor for RNA polymerase I which contains multiple DNA-binding motifs. Among these DNA-binding motifs, HMG box I is essential for promoting RNA polymerase I-dependent rRNA gene transcription. Gel shift assay indicated that the binding of recombinant HMG box I to a 136-bp linear DNA probe was significantly inhibited by Cd2+ at 1 microM. The formation of larger protein-DNA complexes was particularly sensitive to Cd2+. The interaction between HMG box I and DNA was completely inhibited by 10 microM of Cd2+, yet this interaction was not inhibited by the same concentration of Ca2+. Hg2+ at 0.1 microM began to cause abnormal band shifting, and protein-DNA bands disappeared to the wells of a polyacrylamide gel in the presence of 10 microM of Hg2+, reflecting that a drastic change in the conformation of HMG box I-DNA had occurred. The binding of HMG box I to DNA was slightly disturbed by As3+ at 1 microM and was significantly affected at 10 microM. Our results suggest that inhibition of the normal binding of UBF to its target DNA may be one of the mechanisms of heavy metal-induced inhibition of RNA synthesis.

Animals↗
PubMed2001

Molecular evolution of Sry and Sox gene.

The mammalian Sry on the short arm of the Y chromosome encodes a nuclear factor-like protein harboring a DNA-binding domain known as the HMG box. The Sox genes encode similar factor like proteins, but the sequence similarity of the HMG box to that of Sry is variable as being at least 60%. The functional relationship of Sox to Sry genes with special reference to sex determination is unclear except for a few items such as human autosomal Sox9. Thus, it is significant to know more about the evolutionary in addition to the functional relationship between Sry and Sox genes for deepening and broadening our understanding concerning primary sex determination. Therefore, to clarify the ancestry and molecular evolution of the mammalian sex determining gene Sry with its evolutionary relationships to the Sox gene, a molecular phylogenetic tree for the HMG box superfamily was constructed and analyzed, and the following conclusions were reached: (1) The nuclear non histone HMG proteins are supposedly the oldest, appearing at least more than one billion years ago, before the divergence of animals and plants. They diverged into two subgroups: one contains HMG14 and HMG17, and the other one contains HMG1 and HMG2 with various other genes. Subsequent divergences include the nucleolar UBF, nuclear SSRP as well as fungal mating protein Mc, MAT and Ste11. (2) The Sox and Sry genes diverged following the diversification of lymphoid transcription factors TCF and LEF. The Sry gene might have definitely evolved from the Sox gene cluster a few hundred million years ago. Additionally, the marsupial Sry, e.g. from Wallabie's and Dunnart's, is distinguished by being distant from eutherian Sry, but being closely related to the Sox gene cluster. (3) Molecular evolutionary rates estimated in mammalian Sry as the divergent rate per 100 million years are much higher than in Sox genes or other genes from the HMG box superfamily. This rapid evolution of Sry might agree with the fact that the Srys are present not on the pseudoautosomal region but on the distal region with no recombination of the Y chromosomal short arm.

Amino Acid Sequence↗
PubMed2000

The AgNOR proteins: qualitative and quantitative changes during the cell cycle.

AgNOR proteins are a set of argyrophilic nucleolar proteins that accumulate in highly proliferating cells whereas their expression is very low in non-proliferating cells. Some of these proteins remain associated with the nucleolar organizer regions (NORs) during mitosis. In situ, the expression of AgNOR proteins is measured globally by quantification of the level of silver staining using morphometry and image analysis. To go deeper into the understanding of the relationship between the cell cycle and quantity of AgNOR proteins, it was necessary to determine the phases of cell cycle during which expression of AgNOR varies and what are the most variable proteins in each phase. To answer these questions, we set up the protocol permitting to detect and quantify AgNOR proteins on protein samples electrophoresed and transferred onto nitrocellulose membranes. This approach makes it possible to quantitatively evaluate individual AgNOR proteins and identify them, using nucleolar, nuclear and whole interphasic cell extracts, and chromosome-associated protein extracts. By this means, we identified nucleolin and protein B23 as the two major AgNOR proteins in the nucleolus during interphase and subunits of RNA polymerase I and transcription factor UBF as AgNOR proteins remaining associated with NORs during mitosis. We also observed that the increase in the level of nucleolin and protein B23 in rat liver seems to be linked with the cell cycle and not exclusively with stimulation of ribosomal gene (rDNA) transcription. Similarly in synchronized cells, the amount of nucleolin rapidly increases when cells enter the S phase (1.6-fold of the value of serum-deprived cells at 9 h, and 2.35-fold at 12 h after refeeding). The amount of protein B23 exhibits a lower and progressive increase with a maximum when the percentage of cells in G2 phase increased, i.e. after 24 h of cell cycle stimulation. We consider that the amount of AgNOR proteins can be a marker of proliferation, because this amount is related to cell cycle phases, schematically low for G1 phase and high for S-G2 phase. Thus, it is a measure of the relative proportion of cells in each phase, and consequently of the timing of each phase. The higher value indicates that the major part of the cells are in the S-G2 phase and correlatively few are in the G1 phase, and this characterizes a rapid cell cycle.

Animals↗
PubMedDate not supplied

Proteasome-dependent processing of nuclear proteins is correlated with their subnuclear localization.

Although proteasomes are abundant in the nucleoplasm little is known of proteasome-dependent proteolysis within the nucleus. Thus, we monitored the subcellular distribution of nuclear proteins in correlation with proteasomes. The proteasomal pathway clears away endogenous proteins, regulates numerous cellular processes, and delivers immunocompetent peptides to the antigen presenting machinery. Confocal laser scanning microscopy revealed that histones, splicing factor SC35, spliceosomal components, such as U1-70k or SmB/B('), and PML partially colocalize with 20S proteasomes in nucleoplasmic substructures, whereas the centromeric and nucleolar proteins topoisomerase I, fibrillarin, and UBF did not overlap with proteasomes. The specific inhibition of proteasomal processing with lactacystin induced accumulation of histone protein H2A, SC35, spliceosomal components, and PML, suggesting that these proteins are normally degraded by proteasomes. In contrast, concentrations of centromeric proteins CENP-B and -C and nucleolar proteins remained constant during inhibition of proteasomes. Quantification of fluorescence intensities corroborated that nuclear proteins which colocalize with proteasomes are degraded by proteasome-dependent proteolysis within the nucleoplasm. These data provide evidence that the proteasome proteolytic pathway is involved in processing of nuclear components, and thus may play an important role in the regulation of nuclear structure and function.

Acetylcysteine↗
PubMed2001

Molecular mechanisms mediating methylation-dependent silencing of ribosomal gene transcription.

Epigenetic control mechanisms silence about half of ribosomal RNA genes (rDNA) in metabolically active cells. In the mouse, 40% of rDNA repeats are methylated and can be activated by 5-azacytidine treatment. In exploring the effect of methylation on rDNA transcription, we found that methylation of a single CpG dinucleotide within the upstream control element of the rDNA promoter (at -133) abrogates rDNA transcription both in transfection experiments and in in vitro assays using chromatin templates. Chromatin immunoprecipitation assays demonstrate that methylation of the cytosine at -133 inhibits binding of the transcription factor UBF to nucleosomal rDNA, thereby preventing initiation complex formation. Thus, methylation may be a mechanism to inactivate rDNA genes and propagate transcriptional silencing through cell division.

3T3 Cells↗