Click here to close
Hello! We notice that you are using Internet Explorer, which is not supported by Xenbase and may cause the site to display incorrectly.
We suggest using a current version of Chrome,
FireFox, or Safari.
RNA polymerase III transcription in synthetic nuclei assembled in vitro from defined DNA templates.
Ullman KS
,
Forbes DJ
.
???displayArticle.abstract???
Although much is known of the basic control of transcription, little is understood of the way in which the structural organization of the nucleus affects transcription. Synthetic nuclei, assembled de novo in extracts of Xenopus eggs, would be predicted to have a large potential for approaching the role of nuclear structure in RNA biogenesis. Synthetic nuclei provide a system in which the genetic content of the nuclei, as well as the structural and enzymatic proteins within the nuclei, can be manipulated. In this study, we have begun to examine transcription in such nuclei by using the most simple of templates, RNA polymerase III (pol III)-transcribed genes. DNA encoding tRNA or 5S genes was added to an assembly extract, and nuclei were formed entirely from the pol III templates. Conditions which allowed nuclear assembly and pol III transcription to take place efficiently and simultaneously in the assembly extract were found. To examine whether pol III transcription could initiate within synthetic nuclei, or instead was inhibited in nuclei and initiated only on rare unincorporated templates, we identified transcriptional inhibitors that were excluded from nuclei. We found that these inhibitors, heparin and dextran sulfate, blocked pol III transcription in the absence of assembly but did not do so following nuclear assembly. At the concentrations used, the inhibitors had no deleterious effect on nuclear structure itself or on nuclear import. We conclude that pol III transcription is active in synthetic nuclei, and this conclusion is further strengthened by the finding that pol III transcripts could be coisolated with synthetic nuclei. The rapid and direct transcriptional analysis possible with pol III templates, coupled with the simple experimental criteria developed in this study for distinguishing between nuclear and non-nuclear transcription, should now allow a molecular analysis of the effect of nuclear structure on transcriptional and posttranscriptional control.
Adachi,
Study of the cell cycle-dependent assembly of the DNA pre-replication centres in Xenopus egg extracts.
1994, Pubmed,
Xenbase
Adachi,
Study of the cell cycle-dependent assembly of the DNA pre-replication centres in Xenopus egg extracts.
1994,
Pubmed
,
Xenbase
Adeniyi-Jones,
Generation of long read-through transcripts in vivo and in vitro by deletion of 3' termination and processing sequences in the human tRNAimet gene.
1984,
Pubmed
,
Xenbase
Adolph,
Role of nonhistone proteins in metaphase chromosome structure.
1977,
Pubmed
Almouzni,
Replication-coupled chromatin assembly is required for the repression of basal transcription in vivo.
1993,
Pubmed
,
Xenbase
Almouzni,
Nuclear assembly, structure, and function: the use of Xenopus in vitro systems.
1993,
Pubmed
,
Xenbase
Almouzni,
Competition between transcription complex assembly and chromatin assembly on replicating DNA.
1990,
Pubmed
,
Xenbase
Amberg,
Isolation and characterization of RAT1: an essential gene of Saccharomyces cerevisiae required for the efficient nucleocytoplasmic trafficking of mRNA.
1992,
Pubmed
Barton,
The erythroid protein cGATA-1 functions with a stage-specific factor to activate transcription of chromatin-assembled beta-globin genes.
1993,
Pubmed
Barton,
Regulated expression of the beta-globin gene locus in synthetic nuclei.
1994,
Pubmed
,
Xenbase
Bauer,
In vitro assembly of coiled bodies in Xenopus egg extract.
1994,
Pubmed
,
Xenbase
Bell,
In vitro assembly of prenucleolar bodies in Xenopus egg extract.
1992,
Pubmed
,
Xenbase
Blow,
Initiation of DNA replication in nuclei and purified DNA by a cell-free extract of Xenopus eggs.
1986,
Pubmed
,
Xenbase
Carter,
Discrete nuclear domains of poly(A) RNA and their relationship to the functional organization of the nucleus.
1991,
Pubmed
Cox,
DNA replication occurs at discrete sites in pseudonuclei assembled from purified DNA in vitro.
1991,
Pubmed
,
Xenbase
Dabauvalle,
Identification of a soluble precursor complex essential for nuclear pore assembly in vitro.
1990,
Pubmed
,
Xenbase
Dargemont,
Direct interaction of nucleoporin p62 with mRNA during its export from the nucleus.
1995,
Pubmed
,
Xenbase
Dasso,
RCC1, a regulator of mitosis, is essential for DNA replication.
1992,
Pubmed
,
Xenbase
Doye,
A novel nuclear pore protein Nup133p with distinct roles in poly(A)+ RNA transport and nuclear pore distribution.
1994,
Pubmed
Dworetzky,
Translocation of RNA-coated gold particles through the nuclear pores of oocytes.
1988,
Pubmed
,
Xenbase
Elliott,
mRNA nuclear export.
1994,
Pubmed
Fabre,
Nup145p is required for nuclear export of mRNA and binds homopolymeric RNA in vitro via a novel conserved motif.
1994,
Pubmed
Fang,
Evidence that the G1-S and G2-M transitions are controlled by different cdc2 proteins in higher eukaryotes.
1991,
Pubmed
,
Xenbase
Featherstone,
A monoclonal antibody against the nuclear pore complex inhibits nucleocytoplasmic transport of protein and RNA in vivo.
1988,
Pubmed
,
Xenbase
Feldherr,
Role of nuclear trafficking in regulating cellular activity.
1994,
Pubmed
Finlay,
Reconstitution of biochemically altered nuclear pores: transport can be eliminated and restored.
1990,
Pubmed
,
Xenbase
Finlay,
A complex of nuclear pore proteins required for pore function.
1991,
Pubmed
Forbes,
Spontaneous formation of nucleus-like structures around bacteriophage DNA microinjected into Xenopus eggs.
1983,
Pubmed
,
Xenbase
Forbes,
Structure and function of the nuclear pore complex.
1992,
Pubmed
,
Xenbase
Gerace,
Molecular trafficking across the nuclear pore complex.
1992,
Pubmed
Gottesfeld,
Mitotic repression of RNA polymerase III transcription in vitro mediated by phosphorylation of a TFIIIB component.
1994,
Pubmed
,
Xenbase
Hartl,
Mitotic repression of transcription in vitro.
1993,
Pubmed
,
Xenbase
Hartl,
Nuclear assembly with lambda DNA in fractionated Xenopus egg extracts: an unexpected role for glycogen in formation of a higher order chromatin intermediate.
1994,
Pubmed
,
Xenbase
Hassan,
Replication and transcription sites are colocalized in human cells.
1994,
Pubmed
Hirano,
Topoisomerase II does not play a scaffolding role in the organization of mitotic chromosomes assembled in Xenopus egg extracts.
1993,
Pubmed
,
Xenbase
Hozák,
Replication factories.
1994,
Pubmed
Hozák,
Replication factories and nuclear bodies: the ultrastructural characterization of replication sites during the cell cycle.
1994,
Pubmed
Huang,
Nascent pre-mRNA transcripts are associated with nuclear regions enriched in splicing factors.
1991,
Pubmed
Jackson,
The organization of replication centres in higher eukaryotes.
1990,
Pubmed
Jackson,
Visualization of focal sites of transcription within human nuclei.
1993,
Pubmed
Jarmolowski,
Nuclear export of different classes of RNA is mediated by specific factors.
1994,
Pubmed
,
Xenbase
Kadowaki,
Isolation and characterization of Saccharomyces cerevisiae mRNA transport-defective (mtr) mutants.
1994,
Pubmed
Kamakaka,
Potentiation of RNA polymerase II transcription by Gal4-VP16 during but not after DNA replication and chromatin assembly.
1993,
Pubmed
Kassavetis,
Two essential components of the Saccharomyces cerevisiae transcription factor TFIIIB: transcription and DNA-binding properties.
1991,
Pubmed
Kassavetis,
Transcription factor IIIB generates extended DNA interactions in RNA polymerase III transcription complexes on tRNA genes.
1989,
Pubmed
Kornbluth,
Membrane localization of the kinase which phosphorylates p34cdc2 on threonine 14.
1994,
Pubmed
,
Xenbase
Krug,
The regulation of export of mRNA from nucleus to cytoplasm.
1993,
Pubmed
Lang,
Molecular mobility and nucleocytoplasmic flux in hepatoma cells.
1986,
Pubmed
Laskey,
Assembly of the cell nucleus.
1990,
Pubmed
Laybourn,
Threshold phenomena and long-distance activation of transcription by RNA polymerase II.
1992,
Pubmed
Lazarides,
Actin is the naturally occurring inhibitor of deoxyribonuclease I.
1974,
Pubmed
Leno,
The nuclear membrane determines the timing of DNA replication in Xenopus egg extracts.
1991,
Pubmed
,
Xenbase
Lewin,
Chromatin and gene expression: constant questions, but changing answers.
1994,
Pubmed
Logan,
Effects of antibiotics on RNA polymerase III transcription.
1988,
Pubmed
Lohka,
Induction of nuclear envelope breakdown, chromosome condensation, and spindle formation in cell-free extracts.
1985,
Pubmed
,
Xenbase
Lohka,
Roles of cytosol and cytoplasmic particles in nuclear envelope assembly and sperm pronuclear formation in cell-free preparations from amphibian eggs.
1984,
Pubmed
,
Xenbase
Lohka,
Formation in vitro of sperm pronuclei and mitotic chromosomes induced by amphibian ooplasmic components.
1983,
Pubmed
,
Xenbase
Miake-Lye,
Induction of early mitotic events in a cell-free system.
1985,
Pubmed
,
Xenbase
Mills,
Replication occurs at discrete foci spaced throughout nuclei replicating in vitro.
1989,
Pubmed
,
Xenbase
Méchali,
DNA synthesis in a cell-free system from Xenopus eggs: priming and elongation on single-stranded DNA in vitro.
1982,
Pubmed
,
Xenbase
Newmeyer,
Nuclear import can be separated into distinct steps in vitro: nuclear pore binding and translocation.
1988,
Pubmed
,
Xenbase
Newmeyer,
In vitro transport of a fluorescent nuclear protein and exclusion of non-nuclear proteins.
1986,
Pubmed
,
Xenbase
Newmeyer,
Egg extracts for nuclear import and nuclear assembly reactions.
1991,
Pubmed
,
Xenbase
Newmeyer,
Assembly in vitro of nuclei active in nuclear protein transport: ATP is required for nucleoplasmin accumulation.
1986,
Pubmed
,
Xenbase
Newport,
The nucleus: structure, function, and dynamics.
1987,
Pubmed
Newport,
Characterization of the membrane binding and fusion events during nuclear envelope assembly using purified components.
1992,
Pubmed
,
Xenbase
Newport,
A lamin-independent pathway for nuclear envelope assembly.
1990,
Pubmed
,
Xenbase
Newport,
Disassembly of the nucleus in mitotic extracts: membrane vesicularization, lamin disassembly, and chromosome condensation are independent processes.
1987,
Pubmed
,
Xenbase
Newport,
The role of mitotic factors in regulating the timing of the midblastula transition in Xenopus.
1985,
Pubmed
,
Xenbase
Newport,
Nuclear reconstitution in vitro: stages of assembly around protein-free DNA.
1987,
Pubmed
,
Xenbase
Pazin,
ATP-dependent nucleosome reconfiguration and transcriptional activation from preassembled chromatin templates.
1994,
Pubmed
Peterson,
Characterization of two xenopus somatic 5S DNAs and one minor oocyte-specific 5S DNA.
1980,
Pubmed
,
Xenbase
Powers,
Reconstituted nuclei depleted of a vertebrate GLFG nuclear pore protein, p97, import but are defective in nuclear growth and replication.
1995,
Pubmed
,
Xenbase
Schlenstedt,
The GTP-bound form of the yeast Ran/TC4 homologue blocks nuclear protein import and appearance of poly(A)+ RNA in the cytoplasm.
1995,
Pubmed
Sheehan,
Steps in the assembly of replication-competent nuclei in a cell-free system from Xenopus eggs.
1988,
Pubmed
,
Xenbase
Smith,
Domains of the positive transcription factor specific for the Xenopus 5S RNA gene.
1984,
Pubmed
,
Xenbase
Smythe,
Systems for the study of nuclear assembly, DNA replication, and nuclear breakdown in Xenopus laevis egg extracts.
1991,
Pubmed
,
Xenbase
Spector,
Macromolecular domains within the cell nucleus.
1993,
Pubmed
Sullivan,
Calcium mobilization is required for nuclear vesicle fusion in vitro: implications for membrane traffic and IP3 receptor function.
1993,
Pubmed
,
Xenbase
Vigers,
A distinct vesicle population targets membranes and pore complexes to the nuclear envelope in Xenopus eggs.
1991,
Pubmed
,
Xenbase
Wallrath,
Architectural variations of inducible eukaryotic promoters: preset and remodeling chromatin structures.
1994,
Pubmed
Wansink,
Fluorescent labeling of nascent RNA reveals transcription by RNA polymerase II in domains scattered throughout the nucleus.
1993,
Pubmed
Wente,
A temperature-sensitive NUP116 null mutant forms a nuclear envelope seal over the yeast nuclear pore complex thereby blocking nucleocytoplasmic traffic.
1993,
Pubmed
Wolffe,
Differential 5S RNA gene expression in vitro.
1987,
Pubmed
,
Xenbase
Wolffe,
Transcriptional activation of Xenopus class III genes in chromatin isolated from sperm and somatic nuclei.
1989,
Pubmed
,
Xenbase
Wolffe,
Replication timing and Xenopus 5S RNA gene transcription in vitro.
1993,
Pubmed
,
Xenbase
Workman,
Multiple functions of nucleosomes and regulatory factors in transcription.
1993,
Pubmed
Xing,
Higher level organization of individual gene transcription and RNA splicing.
1993,
Pubmed
Yan,
An analysis of the regulation of DNA synthesis by cdk2, Cip1, and licensing factor.
1995,
Pubmed
,
Xenbase
