The cdk7-cyclin H-MAT1 complex associated with TFIIH is localized in coiled bodies.
AuthorsJordan P, Cunha C, Carmo-Fonseca M
JournalMol Biol Cell
PubMed ID9243502
TFIIH is a general transcription factor for RNA polymerase II that in addition is involved in DNA excision repair. TFIIH is composed of eight or nine subunits and we show that at least four of them, namely cdk7, cyclin H, MAT1, and p62 are localized in the coiled body, a ... More
The putative helicase of the coronavirus mouse hepatitis virus is processed from the replicase gene polyprotein and localizes in complexes that are active in viral RNA synthesis.
AuthorsDenison MR, Spaan WJ, van der Meer Y, Gibson CA, Sims AC, Prentice E, Lu XT
JournalJ Virol
PubMed ID10400784
'The coronavirus mouse hepatitis virus (MHV) translates its replicase gene (gene 1) into two co-amino-terminal polyproteins, polyprotein 1a and polyprotein 1ab. The gene 1 polyproteins are processed by viral proteinases to yield at least 15 mature products, including a putative RNA helicase from polyprotein 1ab that is presumed to be ... More
The nucleus: a highly organized but dynamic structure.
AuthorsGonzalez-Melendi P, Beven A, Boudonck K, Abranches R, Wells B, Dolan L, Shaw P
JournalJ Microsc
PubMed ID10849198
'The nucleus in plants and animals is a highly structured organelle containing several well-defined subregions or suborganelles. These include the nucleolus, interphase chromosome territories and coiled bodies. We have visualized transcription sites in plants at both light- and electron-microscopy level by the incorporation of BrUTP. In the nucleolus many dispersed ... More
Replication of hepatitis C virus RNA occurs in a membrane-bound replication complex containing nonstructural viral proteins and RNA.
AuthorsEl-Hage N, Luo G
JournalJ Gen Virol
PubMed ID13679611
'Biochemical studies revealed that nonstructural proteins of hepatitis C virus (HCV) interacted with each other and were associated with intracellular membranes. The goals of this study were to determine whether nonstructural viral proteins are colocalized at specific intracellular sites where HCV RNA is replicated and to identify the virus components ... More
A kinetic framework for a mammalian RNA polymerase in vivo.
AuthorsDundr M, Hoffmann-Rohrer U, Hu Q, Grummt I, Rothblum LI, Phair RD, Misteli T
JournalScience
PubMed ID12446911
'We have analyzed the kinetics of assembly and elongation of the mammalian RNA polymerase I complex on endogenous ribosomal genes in the nuclei of living cells with the use of in vivo microscopy. We show that components of the RNA polymerase I machinery are brought to ribosomal genes as distinct ... More
Alteration of nuclear lamin organization inhibits RNA polymerase II-dependent transcription.
AuthorsSpann TP, Goldman AE, Wang C, Huang S, Goldman RD
JournalJ Cell Biol
PubMed ID11854306
'Regulation of gene activity is mediated by alterations in chromatin organization. In addition, chromatin organization may be governed in part by interactions with structural components of the nucleus. The nuclear lamins comprise the lamina and a variety of nucleoplasmic assemblies that together are major structural components of the nucleus. Furthermore, ... More
Utilizations of various uridine 5'-triphosphate analogues by DNA-dependent RNA polymerases I and II purified from liver nuclei of the cherry salmon (Oncorhynchus masou).
AuthorsNakayama C, Saneyoshi M
JournalJ Biochem (Tokyo)
PubMed ID6526817
'Various 5-substituted UTPs (methyl, ethyl, n-propyl, n-butyl, fluoro, chloro, bromo, and iodo) and sulfur-containing UTP analogues (4-thio-, 2-thio-, 5-methyl-2-thio-, and 5-methyl-4-thio-) were synthesized chemically and their utilization by DNA-dependent-RNA polymerases I and II of the cherry salmon (Oncorhynchus masou) were studied in substitution experiments under the condition of limited RNA ... More
The step-wise assembly of a functional nucleolus in preimplantation mouse embryos involves the cajal (coiled) body.
AuthorsZatsepina O, Baly C, Chebrout M, Debey P
JournalDev Biol
PubMed ID12490198
'After fertilization, ribosomal RNA synthesis is silenced during a period which depends on the species. Data concerning the reassembly of a functional nucleolus remain scarce. We have examined by immunocytochemistry, Western blots, and BrUTP microinjection the dynamics of major nucleolar proteins during the first cycles of mouse embryogenesis, in relation ... More
The transcription cycle of RNA polymerase II in living cells.
AuthorsKimura H, Sugaya K, Cook PR
JournalJ Cell Biol
PubMed ID12473686
'RNA polymerase II transcribes most eukaryotic genes. Its catalytic subunit was tagged with green fluorescent protein and expressed in Chinese hamster cells bearing a mutation in the same subunit; it complemented the defect and so was functional. Photobleaching revealed two kinetic fractions of polymerase in living nuclei: approximately 75% moved ... More
In situ fluorescence visualization of bromouridine incorporated into newly transcribed nucleolar RNA.
'Bromouridine-triphosphate is commonly used for in situ immunocytochemical labeling of newly synthesized RNA in living cells. While extranucleolar transcripts do not require special conditions for visualization, special treatment prior to fixation (e.g. incubation with alpha-amanitine) is necessary for immunofluorescence detection of bromouridine-labeled nucleolar RNA in previous studies. We show in ... More
Nuclear organization studied with the help of a hypotonic shift: its use permits hydrophilic molecules to enter into living cells.
AuthorsKoberna K, Stanek D, Malínský J, Eltsov M, Pliss A, Ctrnáctá V, Cermanová S, Raska I
JournalChromosoma
PubMed ID10525969
'A new procedure for introduction of hydrophilic molecules into living cells based on efficient uptake of these molecules into the cells during hypotonic treatment is presented and its use is demonstrated by a variety of applications. Experiments with cultured vertebrate and Drosophila cells and various animal tissues demonstrated that the ... More
Protein synthesis. Believe it or not-translation in the nucleus.
AuthorsHentze MW
JournalScience
PubMed ID11498567
Semi-conservative transcription of double-stranded RNA catalyzed by bacteriophage phi 6 RNA polymerase.
AuthorsEmori Y, Iba H, Okada Y
JournalJ Biochem (Tokyo)
PubMed ID7462197
Treatment of Pseudomonas phaseolicola double-stranded RNA bacteriophage phi 6 with sodium deoxycholate converted the virions to nucleocapsids, which had in vitro RNA polymerase activity. The incorporation of [3H]UMP continued for at least 7 h. The initial incorporation was detected as intermediate RNA. Radioactivity was chased first into three segments of ... More
Cloning, functional expression and tissue distribution of the human P2Y6 receptor.
AuthorsCommuni D, Parmentier M, Boeynaems JM
JournalBiochem Biophys Res Commun
PubMed ID8670200
In order to isolate new subtypes of P2Y purinoceptors, a human placenta cDNA library was screened at middle stringency with a P2Y4 probe. The purification and the sequencing of several clones led us to identify a 984 base pair open reading frame encoding a new human P2Y receptor. It appeared ... More
Disassembly of interchromatin granule clusters alters the coordination of transcription and pre-mRNA splicing.
AuthorsSacco-Bubulya P, Spector DL
JournalJ Cell Biol
PubMed ID11827980
To examine the involvement of interchromatin granule clusters (IGCs) in transcription and pre-mRNA splicing in mammalian cell nuclei, the serine-arginine (SR) protein kinase cdc2-like kinase (Clk)/STY was used as a tool to manipulate IGC integrity in vivo. Both immunofluorescence and transmission electron microscopy analyses of cells overexpressing Clk/STY indicate that ... More
Targeting of U4/U6 small nuclear RNP assembly factor SART3/p110 to Cajal bodies.
AuthorsStanek D, Rader SD, Klingauf M, Neugebauer KM
JournalJ Cell Biol
PubMed ID12578909
The spliceosomal small nuclear RNAs (snRNAs) are distributed throughout the nucleoplasm and concentrated in nuclear inclusions termed Cajal bodies (CBs). A role for CBs in the metabolism of snRNPs has been proposed but is not well understood. The SART3/p110 protein interacts transiently with the U6 and U4/U6 snRNPs and promotes ... More
Pharmacological characterization of the human P2Y4 receptor.
AuthorsCommuni D, Motte S, Boeynaems JM, Pirotton S
JournalEur J Pharmacol
PubMed ID8997625
The P2Y4 receptor is a new member of the P2Y family which functionally behaves as a pyrimidinergic receptor. The pharmacological properties of the human P2Y4 receptor have been characterized following its stable expression in 1321N1 astrocytoma cells. UTP induced a biphasic accumulation of inositol trisphosphates, with an early peak at ... More
Morpholino oligonucleotide-triggered knockdown reveals a role for maternal E-cadherin during early mouse development.
AuthorsKanzler B, Haas-Assenbaum A, Haas I, Morawiec L, Huber E, Boehm T
JournalMech Dev
PubMed ID14654215
We report that gene silencing via intracytoplasmic microinjections of morpholino-modified antisense oligonucleotides is an effective and reproducible method to study both maternal and zygotic gene functions during early and late stages of mouse preimplantation development. The zygotic expression of the beta-geo transgene in the ROSA26 mouse strain could be inhibited ... More
Ultrastructural analysis of nucleolar transcription in cells microinjected with 5-bromo-UTP.
AuthorsCmarko D, Verschure PJ, Rothblum LI, Hernandez-Verdun D, Amalric F, van Driel R, Fakan S
JournalHistochem Cell Biol
PubMed ID10817672
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 ... More
Acquisition of transcriptional competence in the 1-cell mouse embryo: requirement for recruitment of maternal mRNAs.
AuthorsAoki F, Hara KT, Schultz RM
JournalMol Reprod Dev
PubMed ID12548659
We previously suggested that the transcriptional machinery is rate-limiting for genome activation in the preimplantation mouse embryo (Aoki et al., 1997: Dev Biol 181:296-307), suggesting that genome activation requires the synthesis of some proteins following fertilization. To test this hypothesis, transcriptional activity was measured in 1-cell embryos in which protein ... More
Spatiotemporal sites of DNA replication in macro- and micronuclei of the ciliate Paramecium caudatum.
AuthorsTanaka T, Watanabe T
JournalChromosome Res
PubMed ID12733642
Spatiotemporal sites of DNA replication in macro- and micronuclei of the ciliated protozoan Paramecium caudatum were analyzed by confocal laser scanning microscopy following incorporation of the thymidine analogue BrdU and indirect immunofluorescence. In the macronucleus, replication sites were localized to numerous small domains and scattered throughout the nucleoplasm. This pattern ... More
Initially transcribed sequences strongly affect the extent of abortive initiation by RNA polymerase II.
AuthorsKeene RG, Luse DS
JournalJ Biol Chem
PubMed ID10206958
We investigated transcript initiation and early elongation by RNA polymerase II using templates mismatched between -9 and +3 (bubble templates). Highly purified RNA polymerase II alone was able to initiate transcription specifically on these templates in the presence of dinucleotide primers. The length distribution of abortively initiated RNAs was similar ... More
Hepatitis C virus RNA replication occurs on a detergent-resistant membrane that cofractionates with caveolin-2.
AuthorsShi ST, Lee KJ, Aizaki H, Hwang SB, Lai MM
JournalJ Virol
PubMed ID12634374
The mechanism and machinery of hepatitis C virus (HCV) RNA replication are still poorly understood. In this study, we labeled de novo-synthesized viral RNA in situ with bromouridine triphosphate (BrUTP) in Huh7 cells expressing an HCV subgenomic replicon. By immunofluorescence staining using an anti-BrUTP antibody and confocal microscopy, we showed ... More
Association of phosphatidylinositol 3-kinase with nuclear transcription sites in higher plants.
AuthorsBunney TD, Watkins PA, Beven AF, Shaw PJ, Hernandez LE, Lomonossoff GP, Shanks M, Peart J, Drobak BK
JournalPlant Cell
PubMed ID11006340
The kinases responsible for phosphorylation of inositol-containing lipids are essential for many aspects of normal eukaryotic cell function. Genetic and biochemical studies have established that the phosphatidylinositol (PtdIns) 3-kinase encoded by the yeast VPS34 gene is essential for the efficient sorting and delivery of proteins to the vacuole; the kinase ... More
ORF1a-encoded replicase subunits are involved in the membrane association of the arterivirus replication complex.
Authorsvan der Meer Y, van Tol H, Locker JK, Snijder EJ
JournalJ Virol
PubMed ID9658116
Among the functions of the replicase of equine arteritis virus (EAV; family Arteriviridae, order Nidovirales) are important viral enzyme activities such as proteases and the putative RNA polymerase and RNA helicase functions. The replicase is expressed in the form of two polyproteins (open reading frame 1a [ORF1a] and ORF1ab), which ... More
Relationship between vaccinia virus intracellular cores, early mRNAs, and DNA replication sites.
AuthorsMallardo M, Leithe E, Schleich S, Roos N, Doglio L, Krijnse Locker J
JournalJ Virol
PubMed ID11967332
Virus assembly, a late event in the life cycle of vaccinia virus (VV), is preceded by a number of steps that all occur in the cytoplasm of the infected host cell: virion entry, delivery of the viral core into the cytoplasm, and transcription from these cores of early mRNAs, followed ... More
Nuclear matrix bound fibroblast growth factor receptor is associated with splicing factor rich and transcriptionally active nuclear speckles.
AuthorsSomanathan S, Stachowiak EK, Siegel AJ, Stachowiak MK, Berezney R
JournalJ Cell Biochem
PubMed ID14587039
We have used confocal microscopy combined with computer image analysis to evaluate the functional significance of a constitutively expressed form of the receptor tyrosine kinase FGFR1 (fibroblast growth factor receptor 1) in the nucleus of rapidly proliferating serum stimulated TE 671 cells, a medullobastoma human cell line. Our results demonstrate ... More
Ultrastructural analysis of transcription and splicing in the cell nucleus after bromo-UTP microinjection.
AuthorsCmarko D, Verschure PJ, Martin TE, Dahmus ME, Krause S, Fu XD, van Driel R, Fakan S
JournalMol Biol Cell
PubMed ID9880337
In this study we demonstrate, at an ultrastructural level, the in situ distribution of heterogeneous nuclear RNA transcription sites after microinjection of 5-bromo-UTP (BrUTP) into the cytoplasm of living cells and subsequent postembedding immunoelectron microscopic visualization after different labeling periods. Moreover, immunocytochemical localization of several pre-mRNA transcription and processing factors ... More
Brome mosaic virus RNA replication proteins 1a and 2a colocalize and 1a independently localizes on the yeast endoplasmic reticulum.
AuthorsRestrepo-Hartwig M, Ahlquist P
JournalJ Virol
PubMed ID10559348
The universal membrane association of positive-strand RNA virus RNA replication complexes is implicated in their function, but the intracellular membranes used vary among viruses. Brome mosaic virus (BMV) encodes two mutually interacting RNA replication proteins: 1a, which contains RNA capping and helicase-like domains, and the polymerase-like 2a protein. In cells ... More