scholarly journals Translation of mRNA injected into Xenopus oocytes is specifically inhibited by antisense RNA.

1985 ◽  
Vol 101 (3) ◽  
pp. 1094-1099 ◽  
Author(s):  
R Harland ◽  
H Weintraub
Keyword(s):  
Rna Polymerase ◽  
Antisense Rna ◽  
Xenopus Oocytes ◽  
Oocyte Nucleus ◽  
Detectable Effect ◽  
Antisense Rnas ◽  
Rna Transcripts ◽  
Oocyte Cytoplasm ◽  
Endogenous Proteins ◽  
Specific Mrna

The bacteriophage SP6 promoter and RNA polymerase were used to synthesize sense and antisense RNAs coding for the enzymes thymidine kinase (TK) and chloramphenicol acetyl transferase (CAT). Injection of antisense CAT RNA into frog oocytes inhibited expression of sense CAT mRNA. Similarly, antisense TK RNA inhibited expression of sense TK mRNA. Antisense RNAs were stable in oocytes and had no detectable effect on either the expression of endogenous proteins or on the expression of nonhomologous RNA transcripts. CAT activity expressed from a plasmid transcribed in the oocyte nucleus was also inhibited by antisense RNA injected into the oocyte cytoplasm. The data suggest that antisense RNA will be useful in identifying the function of specific mRNA sequences during early development of the frog.

scholarly journals RNA Polymerase III in Cajal Bodies and Lampbrush Chromosomes of the Xenopus Oocyte Nucleus

2002 ◽  
Vol 13 (10) ◽  
pp. 3466-3476 ◽  
Author(s):  
Christine Murphy ◽  
Zhengxin Wang ◽  
Robert G. Roeder ◽  
Joseph G. Gall
Keyword(s):  
Transcription Factors ◽  
Rna Polymerase ◽  
Rna Polymerase Iii ◽  
Cajal Bodies ◽  
Oocyte Nucleus ◽  
Xenopus Laevis Oocyte ◽  
Lampbrush Chromosomes ◽  
Oocyte Cytoplasm ◽  
Pol Iii

We used immunofluorescence to study the distribution and targeting of RNA polymerase (pol) III subunits and pol III transcription factors in the Xenopus laevis oocyte nucleus. Antibodies against several of these proteins stained Cajal bodies and ∼90 specific sites on the lampbrush chromosomes. Some of the chromosomal sites had been identified previously by in situ hybridization as the genes for 5S rRNA. The remaining sites presumably encode tRNAs and other pol III transcripts. Pol III sites were often resolvable as loops similar to the much more abundant pol II loops, but without a matrix detectable by phase contrast or differential interference contrast. This morphology is consistent with the transcription of short repeated sequences. Hemagglutinin-tagged transcripts encoding core subunits and transcription factors were injected into the oocyte cytoplasm, and the distribution of newly translated proteins inside the nucleus was monitored by immunostaining. Cajal bodies were preferentially targeted by these proteins, and in some cases the chromosomal sites were also weakly stained. The existence of pol III subunits and pol III transcription factors in Cajal bodies and their targeting to these organelles are consistent with a model of Cajal bodies as sites for preassembly of the nuclear transcription machinery.

scholarly journals Mono Q chromatography permits recycling of DNA template and purification of RNA transcripts after T7 RNA polymerase reaction

1991 ◽  
Vol 19 (10) ◽  
pp. 2786-2786 ◽  
Author(s):  
M. J. Jahn ◽  
D. Jahn ◽  
A.M. Kumar ◽  
D. Soll
Keyword(s):  
Rna Polymerase ◽  
T7 Rna Polymerase ◽  
Rna Transcripts ◽  
Dna Template

RNA polymerase activity in virions from Ustilago maydis

1984 ◽  
Vol 4 (1) ◽  
pp. 188-194
Author(s):  
B S Ben-Tzvi ◽  
Y Koltin ◽  
M Mevarech ◽  
A Tamarkin
Keyword(s):  
Rna Polymerase ◽  
Viral Genome ◽  
Ustilago Maydis ◽  
Polymerase Activity ◽  
Reaction Products ◽  
Double Stranded Rna ◽  
Rna Molecules ◽  
Rna Transcripts ◽  
Rna Polymerase Activity

RNA polymerase activity is associated with the double-stranded RNA virions of Ustilago maydis. The reaction products of the polymerase activity are single-stranded RNA molecules. The RNA molecules synthesized are homologous to the three classes of double-stranded RNA molecules that typify the viral genome. The single-stranded RNA synthesized is released from the virions. The molecular weight of the single-stranded RNA transcripts is about half the size of the double-stranded RNA segments, and thus, it appears that in the in vitro reaction, full-length transcripts can be obtained.

scholarly journals The Swi/Snf Chromatin Remodeling Complex Is Required for Ribosomal DNA and Telomeric Silencing in Saccharomyces cerevisiae

2004 ◽  
Vol 24 (18) ◽  
pp. 8227-8235 ◽  
Author(s):  
Vardit Dror ◽  
Fred Winston
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Chromatin Remodeling ◽  
Ribosomal Dna ◽  
Transcriptional Activation ◽  
Rdna Locus ◽  
Detectable Effect ◽  
Chromatin Remodeling Complex ◽  
Two Factors

ABSTRACT The Swi/Snf chromatin remodeling complex has been previously demonstrated to be required for transcriptional activation and repression of a subset of genes in Saccharomyces cerevisiae. In this work we demonstrate that Swi/Snf is also required for repression of RNA polymerase II-dependent transcription in the ribosomal DNA (rDNA) locus (rDNA silencing). This repression appears to be independent of both Sir2 and Set1, two factors known to be required for rDNA silencing. In contrast to many other rDNA silencing mutants that have elevated levels of rDNA recombination, snf2Δ mutants have a significantly decreased level of rDNA recombination. Additional studies have demonstrated that Swi/Snf is also required for silencing of genes near telomeres while having no detectable effect on silencing of HML or HMR.

Injected nuclei in frog oocytes: fate, enlargement, and chromatin dispersal

Development ◽  
1976 ◽  
Vol 36 (3) ◽  
pp. 523-540
Author(s):  
J. B. Gurdon
Keyword(s):  
Bovine Serum Albumin ◽  
Xenopus Laevis ◽  
Serum Albumin ◽  
Bovine Serum ◽  
Oocyte Nucleus ◽  
Oocyte Cytoplasm ◽  
Nuclear Injection

A method is described by which nuclei associated with some cytoplasm can be rapidly prepared from a suspension of cells. The method involves the use of lysolecithin and bovine serum albumin. Oocytes of Xenopus laevis were injected with about 200 nuclei prepared from human He La cells by this method. Nuclei were deposited in oocyte cytoplasm, in the oocyte nucleus, or in the dispersed contents of a ruptured oocyte nucleus. Injected He La nuclei enlarge up to several hundred times in volume in the course of a few days. Their enlargement is associated with chromatin dispersion, increased binding of an acidic dye, and with the reduction in size, and eventual disappearance, of nucleoli. The amount of He La nucleus enlargement is much greater when the oocyte nucleus is ruptured. The fate of injected nuclei was followed by the use of HeLa nuclei whose DNA had been previously la belled with [3H] thymidine. La belled DNA does not pass from injected He La nuclei into the oocyte nucleus. Injected nuclei appear not to fuse with each other or with the oocyte nucleus. Nuclei prepared by the above method look morphologically healthy in oocytes cultured in vitro for up to one month after nuclear injection. Nuclei prepared by other methods, such as those involving the use of detergents, undergo deterioration within a few days after injection into oocytes.

Microinjection of fos-specific antibodies blocks DNA synthesis in fibroblast cells

1988 ◽  
Vol 8 (4) ◽  
pp. 1670-1676 ◽  
Author(s):  
K T Riabowol ◽  
R J Vosatka ◽  
E B Ziff ◽  
N J Lamb ◽  
J R Feramisco
Keyword(s):  
Cell Cycle ◽  
Dna Synthesis ◽  
Antisense Rna ◽  
Proliferating Cells ◽  
Rna Transcripts ◽  
Serum Stimulation ◽  
Inhibition Of Growth ◽  
Nonspecific Immunoglobulins ◽  
3T3 Cell Lines ◽  
Stimulation Of

Transcription of the protooncogene c-fos is increased greater than 10-fold within minutes of treatment of fibroblasts with serum or purified growth factors. Recent experiments with mouse 3T3 cell lines containing inducible fos antisense RNA constructs have shown that induced fos antisense RNA transcripts cause either a marked inhibition of growth in continuously proliferating cells or, conversely, a minimal effect except during the transition from a quiescent (G0) state into the cell cycle. Since intracellular production of large amounts of antisense RNA does not completely block gene expression, we microinjected affinity-purified antibodies raised against fos to determine whether and when during the cell cycle c-fos expression was required for cell proliferation. Using this independent method, we found that microinjected fos antibodies efficiently blocked serum-stimulated DNA synthesis when injected up to 6 to 8 h after serum stimulation of quiescent REF-52 fibroblasts. Furthermore, when fos antibodies were injected into asynchronously growing cells, a consistently greater number of cells was prevented from synthesizing DNA than when cells were injected with nonspecific immunoglobulins. Thus, whereas the activity of c-fos may be necessary for transition of fibroblasts from G0 to G1 of the cell cycle, its function is also required during the early G1 portion of the cell cycle to allow subsequent DNA synthesis.

Identification of Integrator-PP2A complex (INTAC), an RNA polymerase II phosphatase

Science ◽  
2020 ◽  
Vol 370 (6520) ◽  
pp. eabb5872
Author(s):  
Hai Zheng ◽  
Yilun Qi ◽  
Shibin Hu ◽  
Xuan Cao ◽  
Congling Xu ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Protein Phosphatase ◽  
Rna Cleavage ◽  
Rna Transcripts ◽  
Core Enzyme ◽  
Repeat Domain ◽  
Phosphatase 2A ◽  
Carboxy Terminal ◽  
Resolution Structure

The 14-subunit metazoan-specific Integrator contains an endonuclease that cleaves nascent RNA transcripts. Here, we identified a complex containing Integrator and protein phosphatase 2A core enzyme (PP2A-AC), termed INTAC. The 3.5-angstrom-resolution structure reveals that nine human Integrator subunits and PP2A-AC assemble into a cruciform-shaped central scaffold formed by the backbone and shoulder modules, with the phosphatase and endonuclease modules flanking the opposite sides. As a noncanonical PP2A holoenzyme, the INTAC complex dephosphorylates the carboxy-terminal repeat domain of RNA polymerase II at serine-2, -5, and -7 and thus regulates transcription. Our study extends the function of PP2A to transcriptional regulation and reveals how dual enzymatic activities—RNA cleavage and RNA polymerase II dephosphorylation—are structurally and functionally integrated into the INTAC complex.

scholarly journals DIRS retrotransposons amplify via linear, single-stranded cDNA intermediates

2020 ◽  
Vol 48 (8) ◽  
pp. 4230-4243 ◽  
Author(s):  
Marek Malicki ◽  
Thomas Spaller ◽  
Thomas Winckler ◽  
Christian Hammann
Keyword(s):  
Rna Interference ◽  
Rna Polymerase ◽  
Dictyostelium Discoideum ◽  
Repeat Sequence ◽  
Marker Genes ◽  
Argonaute Protein ◽  
Tyrosine Recombinase ◽  
Rna Dependent Rna Polymerase ◽  
Rna Transcripts ◽  
Complementary Region

Abstract The Dictyostelium Intermediate Repeat Sequence 1 (DIRS-1) is the name-giving member of the DIRS order of tyrosine recombinase retrotransposons. In Dictyostelium discoideum, DIRS-1 is highly amplified and enriched in heterochromatic centromers of the D. discoideum genome. We show here that DIRS-1 it tightly controlled by the D. discoideum RNA interference machinery and is only mobilized in mutants lacking either the RNA dependent RNA polymerase RrpC or the Argonaute protein AgnA. DIRS retrotransposons contain an internal complementary region (ICR) that is thought to be required to reconstitute a full-length element from incomplete RNA transcripts. Using different versions of D. discoideum DIRS-1 equipped with retrotransposition marker genes, we show experimentally that the ICR is in fact essential to complete retrotransposition. We further show that DIRS-1 produces a mixture of single-stranded, mostly linear extrachromosomal cDNA intermediates. If this cDNA is isolated and transformed into D. discoideum cells, it can be used by DIRS-1 proteins to complete productive retrotransposition. This work provides the first experimental evidence to propose a general retrotransposition mechanism of the class of DIRS like tyrosine recombinase retrotransposons.

scholarly journals Splicing Speckles Are Not Reservoirs of RNA Polymerase II, but Contain an Inactive Form, Phosphorylated on Serine2 Residues of the C-Terminal Domain

2006 ◽  
Vol 17 (4) ◽  
pp. 1723-1733 ◽  
Author(s):  
Sheila Q. Xie ◽  
Sonya Martin ◽  
Pascale V. Guillot ◽  
David L. Bentley ◽  
Ana Pombo
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Optical Resolution ◽  
Polymerase Activity ◽  
Terminal Domain ◽  
Transcriptional Inhibition ◽  
Inactive Form ◽  
Splicing Speckles ◽  
Nuclear Domains ◽  
Specific Mrna

“Splicing speckles” are major nuclear domains rich in components of the splicing machinery and polyA+ RNA. Although speckles contain little detectable transcriptional activity, they are found preferentially associated with specific mRNA-coding genes and gene-rich R bands, and they accumulate some unspliced pre-mRNAs. RNA polymerase II transcribes mRNAs and is required for splicing, with some reports suggesting that the inactive complexes are stored in splicing speckles. Using ultrathin cryosections to improve optical resolution and preserve nuclear structure, we find that all forms of polymerase II are present, but not enriched, within speckles. Inhibition of polymerase activity shows that speckles do not act as major storage sites for inactive polymerase II complexes but that they contain a stable pool of polymerase II phosphorylated on serine2 residues of the C-terminal domain, which is transcriptionally inactive and may have roles in spliceosome assembly or posttranscriptional splicing of pre-mRNAs. Paraspeckle domains lie adjacent to speckles, but little is known about their protein content or putative roles in the expression of the speckle-associated genes. We find that paraspeckles are transcriptionally inactive but contain polymerase II, which remains stably associated upon transcriptional inhibition, when paraspeckles reorganize around nucleoli in the form of caps.

scholarly journals Presence and distribution of human papillomavirus sense and antisense RNA transcripts in genital cancers

1991 ◽  
Vol 72 (4) ◽  
pp. 885-895 ◽  
Author(s):  
G. D. Higgins ◽  
D. M. Uzelin ◽  
G. E. Phillips ◽  
C. J. Burrell
Keyword(s):  
Human Papillomavirus ◽  
Antisense Rna ◽  
Rna Transcripts
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