Distinguishing the sites of pre-rRNA synthesis and accumulation in Ehrlich tumor cell nucleoli

1991 ◽  
Vol 99 (4) ◽  
pp. 759-767
Author(s):  
M. Thiry ◽  
G. Goessens
Keyword(s):  
Tumor Cell ◽  
Condensed Chromatin ◽  
Rrna Genes ◽  
Rrna Synthesis ◽  
Rrna Gene ◽  
Dense Fibrillar Component ◽  
Ehrlich Tumor ◽  
Granular Component ◽  
Fibrillar Component

The precise location of transcribing rRNA genes within Ehrlich tumor cell nucleoli has been investigated using two approaches: high-resolution autoradiography of cells pulse-labelled with tritiated uridine, varying the exposure time, and in situ-in vitro transcription coupled with an immunogold labelling procedure. When autoradiographic preparations are exposed for a short time, silver grains are found associated almost exclusively with interphasic cell nucleoli. Labelling of extranucleolar areas requires longer exposure. Within the nucleolus, the first sites to be revealed are in the dense fibrillar component. Prolonging exposure increases labelling over the dense fibrillar component, with label becoming more and more apparent over the fibrillar centers. Under these conditions, however, labelling does not extend into the granular component, and no background is observed. Initiation of transcription on ultrathin cell sections occurs preferentially at the borders of condensed chromatin blocks and in their close vicinity. The condensed chromatin areas themselves remain unlabelled. Inside most nucleoli, gold-particle clusters are mainly detected in the fibrillar centers, especially at their periphery, whereas the dense fibrillar component and the granular component remain devoid of label. These results, together with previous observations made on the same cell type, clearly indicate that the fibrillar centers are the sites of rRNA gene transcription in Ehrlich tumor cell nucleoli, while the dense fibrillar component is the site of pre-rRNA accumulation.

scholarly journals UBF levels determine the number of active ribosomal RNA genes in mammals

2008 ◽  
Vol 183 (7) ◽  
pp. 1259-1274 ◽  
Author(s):  
Elaine Sanij ◽  
Gretchen Poortinga ◽  
Kerith Sharkey ◽  
Sandy Hung ◽  
Timothy P. Holloway ◽  
...  
Keyword(s):  
Ribosomal Rna ◽  
High Mobility ◽  
Rrna Genes ◽  
Rrna Synthesis ◽  
Rrna Gene ◽  
Extracellular Signal ◽  
Binding Factor ◽  
Upstream Binding Factor ◽  
Polymerase I

In mammals, the mechanisms regulating the number of active copies of the ∼200 ribosomal RNA (rRNA) genes transcribed by RNA polymerase I are unclear. We demonstrate that depletion of the transcription factor upstream binding factor (UBF) leads to the stable and reversible methylation-independent silencing of rRNA genes by promoting histone H1–induced assembly of transcriptionally inactive chromatin. Chromatin remodeling is abrogated by the mutation of an extracellular signal-regulated kinase site within the high mobility group box 1 domain of UBF1, which is required for its ability to bend and loop DNA in vitro. Surprisingly, rRNA gene silencing does not reduce net rRNA synthesis as transcription from remaining active genes is increased. We also show that the active rRNA gene pool is not static but decreases during differentiation, correlating with diminished UBF expression. Thus, UBF1 levels regulate active rRNA gene chromatin during growth and differentiation.

scholarly journals Building an efficient factory

2002 ◽  
Vol 157 (5) ◽  
pp. 739-741 ◽  
Author(s):  
Sui Huang
Keyword(s):  
Rrna Synthesis ◽  
Dense Fibrillar Component ◽  
Rdna Transcription ◽  
Granular Component ◽  
Fibrillar Center ◽  
Pol I ◽  
Fibrillar Component ◽  
Polymerase I

The subnucleolar structure that is involved in rDNA transcription has been controversial. A report by Koberna et al. (2002)(this issue, page 743) adds significant weight toward the idea that dense fibrillar components (DFCs)**Abbreviations used in this paper: DFC, dense fibrillar component; FC, fibrillar center; GC, granular component; Pol I, polymerase I. and fibrillar center (FC)/DFC borders are the sites of pre-rRNA synthesis.

scholarly journals Simultaneous immunoelectron microscopic visualization of protein B23 and C23 distribution in the HeLa cell nucleolus.

1989 ◽  
Vol 37 (9) ◽  
pp. 1371-1374 ◽  
Author(s):  
M Biggiogera ◽  
S Fakan ◽  
S H Kaufmann ◽  
A Black ◽  
J H Shaper ◽  
...  
Keyword(s):  
Hela Cell ◽  
Immunoelectron Microscopy ◽  
Silver Staining ◽  
Dense Fibrillar Component ◽  
Specific Antibodies ◽  
Granular Component ◽  
Protein B23 ◽  
Reported Data ◽  
Fibrillar Component

The intranucleolar distribution of phosphoproteins B23 and C23 was visualized simultaneously by post-embedding immunoelectron microscopy in HeLa cell nucleoli, using specific antibodies. The data show that proteins B23 and C23 co-localize to the same nucleolar compartments, i.e., the dense fibrillar component and the granular component. Neither of the two antibodies is significantly associated with the fibrillar centers in these cells, although the fibrillar centers appear positive after silver staining. These findings suggest that other unidentified components must be responsible for the silver staining observed in the fibrillar centers of interphase nucleoli. The results are discussed in the light of previously reported data obtained by preembedding immunolabeling techniques and by silver staining, which both suggested a localization of protein C23 inside the fibrillar centers.

scholarly journals SUMO-1 Modification Alters ADAR1 Editing Activity

2005 ◽  
Vol 16 (11) ◽  
pp. 5115-5126 ◽  
Author(s):  
Joana M.P. Desterro ◽  
Liam P. Keegan ◽  
Ellis Jaffray ◽  
Ron T. Hay ◽  
Mary A. O'Connell ◽  
...  
Keyword(s):  
Rna Editing ◽  
Nucleolar Localization ◽  
Dense Fibrillar Component ◽  
Wild Type ◽  
Fibrillar Center ◽  
Editing Activity ◽  
Fibrillar Component ◽  
Editing Enzyme

We identify ADAR1, an RNA-editing enzyme with transient nucleolar localization, as a novel substrate for sumoylation. We show that ADAR1 colocalizes with SUMO-1 in a subnucleolar region that is distinct from the fibrillar center, the dense fibrillar component, and the granular component. Our results further show that human ADAR1 is modified by SUMO-1 on lysine residue 418. An arginine substitution of K418 abolishes SUMO-1 conjugation and although it does not interfere with ADAR1 proper localization, it stimulates the ability of the enzyme to edit RNA both in vivo and in vitro. Moreover, modification of wild-type recombinant ADAR1 by SUMO-1 reduces the editing activity of the enzyme in vitro. Taken together these data suggest a novel role for sumoylation in regulating RNA-editing activity.

scholarly journals In Exponentially Growing Saccharomyces cerevisiae Cells, rRNA Synthesis Is Determined by the Summed RNA Polymerase I Loading Rate Rather than by the Number of Active Genes

2003 ◽  
Vol 23 (5) ◽  
pp. 1558-1568 ◽  
Author(s):  
Sarah L. French ◽  
Yvonne N. Osheim ◽  
Francesco Cioci ◽  
Masayasu Nomura ◽  
Ann L. Beyer
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Exponential Growth ◽  
Rrna Genes ◽  
Rrna Synthesis ◽  
Rrna Gene ◽  
Transcription Rate ◽  
Initiation Rate ◽  
Pol I ◽  
Genes Encoding ◽  
Active Genes

ABSTRACT Genes encoding rRNA are multicopy and thus could be regulated by changing the number of active genes or by changing the transcription rate per gene. We tested the hypothesis that the number of open genes is limiting rRNA synthesis by using an electron microscopy method that allows direct counting of the number of active genes per nucleolus and the number of polymerases per active gene. Two strains of Saccharomyces cerevisiae were analyzed during exponential growth: a control strain with a typical number of rRNA genes (∼143 in this case) and a strain in which the rRNA gene number was reduced to ∼42 but which grows as well as controls. In control strains, somewhat more than half of the genes were active and the mean number of polymerases/gene was ∼50 ± 20. In the 42-copy strain, all rRNA genes were active with a mean number of 100 ± 29 polymerases/gene. Thus, an equivalent number of polymerases was active per nucleolus in the two strains, though the number of active genes varied by twofold, showing that overall initiation rate, and not the number of active genes, determines rRNA transcription rate during exponential growth in yeast. Results also allow an estimate of elongation rate of ∼60 nucleotides/s for yeast Pol I and a reinitiation rate of less than 1 s on the most heavily transcribed genes.

scholarly journals Genetic relationships of Corynebacterium diphtheriae strains isolated from a diphtheria case and carriers by restriction fragment length polymorphism of rRNA genes

1995 ◽  
Vol 37 (4) ◽  
pp. 291-296
Author(s):  
Claudio Tavares Sacchi ◽  
Ana Paula Silva de Lemos ◽  
Silvana Tadeu Casagrande ◽  
Alice Massumi Mori ◽  
Carmecy Lopes de Almeida
Keyword(s):  
Cell Culture ◽  
Genetic Relationships ◽  
Culture Method ◽  
Vero Cells ◽  
Corynebacterium Diphtheriae ◽  
Rrna Genes ◽  
Diffusion Method ◽  
Rrna Gene

In the present study we report the results of an analysis, based on ribotyping of Corynebacterium diphtheriae intermedius strains isolated from a 9 years old child with clinical diphtheria and his 5 contacts. Quantitative analysis of RFLPs of rRNA was used to determine relatedness of these 7 C.diphtheriae strains providing support data in the diphtheria epidemiology. We have also tested those strains for toxigenicity in vitro by using the Elek's gel diffusion method and in vivo by using cell culture method on cultured monkey kidney cell (VERO cells). The hybridization results revealed that the 5 C.diphtheriae strains isolated from contacts and one isolated from the clinical case (nose case strain) had identical RFLP patterns with all 4 restriction endonucleases used, ribotype B. The genetic distance from this ribotype and ribotype A (throat case strain), that we initially assumed to be responsible for the illness of the patient, was of 0.450 showing poor genetic correlation among these two ribotypes. We found no significant differences concerned to the toxin production by using the cell culture method. In conclusion, the use of RFLPs of rRNA gene was successful in detecting minor differences in closely related toxigenic C.diphtheriae intermedius strains and providing information about genetic relationships among them.

Processing of truncated mouse or human rRNA transcribed from ribosomal minigenes transfected into mouse cells

1994 ◽  
Vol 14 (6) ◽  
pp. 4044-4056
Author(s):  
K V Hadjiolova ◽  
A Normann ◽  
J Cavaillé ◽  
E Soupène ◽  
S Mazan ◽  
...  
Keyword(s):  
18S Rrna ◽  
Processing System ◽  
Rrna Genes ◽  
In Vitro Assays ◽  
Rrna Gene ◽  
28S Rrna ◽  
Rrna Processing ◽  
Mouse Cells

The processing of pre-rRNA in eukaryotic cells involves a complex pattern of nucleolytic reactions taking place in preribosomes with the participation of several nonribosomal proteins and small nuclear RNAs. The mechanism of these reactions remains largely unknown, mainly because of the absence of faithful in vitro assays for most processing steps. We have developed a pre-rRNA processing system using the transient expression of ribosomal minigenes transfected into cultured mouse cells. Truncated mouse or human rRNA genes are faithfully transcribed under the control of mouse promoter and terminator signals. The fate of these transcripts is analyzed by the use of reporter sequences flanking the rRNA gene inserts. Both mouse and human transcripts, containing the 3' end of 18S rRNA-encoding DNA (rDNA), internal transcribed spacer (ITS) 1, 5.8S rDNA, ITS 2, and the 5' end of 28S rDNA, are processed predominantly to molecules coterminal with the natural mature rRNAs plus minor products corresponding to cleavages within ITS 1 and ITS 2. To delineate cis-acting signals in pre-rRNA processing, we studied series of more truncated human-mouse minigenes. A faithful processing at the 18S rRNA/ITS 1 junction can be observed with transcripts containing only the 60 3'-terminal nucleotides of 18S rRNA and the 533 proximal nucleotides of ITS 1. However, further truncation of 18S rRNA (to 8 nucleotides) or of ITS 1 (to 48 nucleotides) abolishes the cleavage of the transcript. Processing at the ITS 2/28S rRNA junction is observed with truncated transcripts lacking the 5.8S rRNA plus a major part of ITS 2 and containing only 502 nucleotides of 28S rRNA. However, further truncation of the 28S rRNA segment to 217 nucleotides abolishes processing. Minigene transcripts containing most internal sequences of either ITS 1 or ITS 2, but devoid of ITS/mature rRNA junctions, are not processed, suggesting that the cleavages in vivo within either ITS segment are dependent on the presence in cis of mature rRNA sequences. These results show that the major cis signals for pre-rRNA processing at the 18S rRNA/ITS 1 or the ITS2/28S rRNA junction involve solely a limited critical length of the respective mature rRNA and adjacent spacer sequences.

Purification and characterization of a high-mobility-group-like DNA-binding protein that stimulates rRNA synthesis in vitro

1988 ◽  
Vol 8 (8) ◽  
pp. 3406-3414
Author(s):  
H F Yang-Yen ◽  
L I Rothblum
Keyword(s):  
Dna Binding ◽  
Binding Protein ◽  
High Mobility ◽  
Dna Binding Protein ◽  
High Mobility Group ◽  
Rrna Synthesis ◽  
Rrna Gene ◽  
Hmg Proteins ◽  
Novikoff Hepatoma

A 16,000-dalton, high-mobility-group-like (HMG-like) DNA-binding protein, referred to as p16, has been purified to homogeneity from Novikoff hepatoma ascites cells. p16 binds specifically to a portion of the 5' flanking region of the rat rRNA gene (-620 to -417), which is part of the upstream activator sequence identified previously (B. G. Cassidy, H.-F. Yang-Yen, and L. I. Rothblum, Mol. Cell. Biol. 6:2766-2773, 1986). p16 also binds to a segment of the external transcribed spacer (+352 to +545). In vitro reconstituted transcription experiments demonstrated that the addition of p16 stimulated rRNA synthesis up to ca. fourfold. The stimulation was dose dependent and saturable. The effect of p16 on ribosomal gene transcription was also dependent on the presence of either the upstream or the downstream DNA-binding site, or both. The amino acid composition of p16 is very similar to that of HMG-I, suggesting that p16 may be a member of the HMG-I family of proteins. In this case, our results suggest that HMG proteins may play an important role in the regulation of the rRNA gene expression.

scholarly journals In vivo regulation of rRNA transcription occurs rapidly in nondividing and dividing Drosophila cells in response to a phorbol ester and serum.

1993 ◽  
Vol 13 (2) ◽  
pp. 928-933 ◽  
Author(s):  
S M Vallett ◽  
M Brudnak ◽  
M Pellegrini ◽  
H W Weber
Keyword(s):  
Phorbol Ester ◽  
Growth Regulation ◽  
Rrna Genes ◽  
Rrna Synthesis ◽  
Rrna Gene ◽  
Rdna Transcription ◽  
Transcription System ◽  
Pol I ◽  
Drosophila Cells

The synthesis of ribosomes is an essential cellular process which requires the transcription of the rRNA genes by RNA polymerase I (Pol I). The regulation of rRNA synthesis is known to be coupled to growth regulation. In nongrowing, slowly growing, and rapidly growing Drosophila cells, exposure to the tumor-promoting phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) increases the synthesis of precursor and mature rRNAs. Using nuclear run-on assays, we show that TPA enhances transcription of the rRNA genes. These results suggest that TPA regulates expression of RNA genes transcribed by Pol I, irrespective of the growth state of the cells. In slowly dividing Drosophila cells, increasing the serum concentration rapidly alters the accumulation of rRNA by enhancing rDNA transcription within 1 h. Thus, TPA and serum are each able to rapidly regulate rRNA gene expression in Drosophila cells. These results indicate that the RNA Pol I transcription system can be regulated by agents which have previously been shown to effect specific genes transcribed by the RNA Pol II system.

scholarly journals Detection and subtype identification of Blastocystis isolates from wastewater samples in the Philippines

2011 ◽  
Vol 9 (1) ◽  
pp. 128-137 ◽  
Author(s):  
Jan Ervin G. Banaticla ◽  
Windell L. Rivera
Keyword(s):  
Wastewater Treatment Plants ◽  
Small Subunit ◽  
The Philippines ◽  
Rrna Genes ◽  
Bootstrap Support ◽  
In Vitro Cultivation ◽  
Rrna Gene ◽  
Ssu Rrna ◽  
Wastewater Samples

To provide further evidence of waterborne transmission of Blastocystis, a total of 31 wastewater treatment plants from geographically distinct locations across the Philippines were sampled for influent and effluent sewage samples. In vitro cultivation was the method of choice to increase sensitivity of detection. Blastocystis cysts were detected in 15% (9/62) of the samples using in vitro culture. Moreover, influent and effluent samples were 23% (7/31) and 7% (2/31) positive for the parasite, respectively. The presence of viable cysts in treated samples may be an indication of the inefficiency of the treatment process in preventing Blastocystis from entering the environment. Polymerase chain reaction and sequencing of the full-length small subunit ribosomal RNA (SSU rRNA) genes of the nine wastewater isolates were performed. The SSU rRNA gene sequences of the isolates showed very high similarity (98 to 99%) to homologous sequences of Blastocystis described previously. The phylogenetic tree constructed showed that the wastewater isolates clustered with each other with good bootstrap support and belonged to two subtypes (ST) – ST1 and ST2. This is the first report of subtyping Blastocystis isolates from wastewater samples and gives further emphasis to the remarkable genetic diversity of the parasite.

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