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Journal ArticleDOI

Accurate transcription initiation by RNA polymerase II in a soluble extract from isolated mammalian nuclei

11 Mar 1983-Nucleic Acids Research (Oxford University Press)-Vol. 11, Iss: 5, pp 1475-1489
TL;DR: A procedure for preparing extracts from nuclei of human tissue culture cells that directs accurate transcription initiation in vitro from class II promoters, including tRNA and Ad 2 VA, is developed.
Abstract: We have developed a procedure for preparing extracts from nuclei of human tissue culture cells that directs accurate transcription initiation in vitro from class II promoters. Conditions of extraction and assay have been optimized for maximum activity using the major late promoter of adenovirus 2. The extract also directs accurate transcription initiation from other adenovirus promoters and cellular promoters. The extract also directs accurate transcription initiation from class III promoters (tRNA and Ad 2 VA).

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Citations
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Journal ArticleDOI
TL;DR: It is shown that micromolar concentrations of H2O2 can induce the expression and replication of HIV‐1 in a human T cell line and suggests that diverse agents thought to activate NF‐kappa B by distinct intracellular pathways might all act through a common mechanism involving the synthesis of ROI.
Abstract: Hydrogen peroxide and oxygen radicals are agents commonly produced during inflammatory processes. In this study, we show that micromolar concentrations of H2O2 can induce the expression and replication of HIV-1 in a human T cell line. The effect is mediated by the NF-kappa B transcription factor which is potently and rapidly activated by an H2O2 treatment of cells from its inactive cytoplasmic form. N-acetyl-L-cysteine (NAC), a well characterized antioxidant which counteracts the effects of reactive oxygen intermediates (ROI) in living cells, prevented the activation of NF-kappa B by H2O2. NAC and other thiol compounds also blocked the activation of NF-kappa B by cycloheximide, double-stranded RNA, calcium ionophore, TNF-alpha, active phorbol ester, interleukin-1, lipopolysaccharide and lectin. This suggests that diverse agents thought to activate NF-kappa B by distinct intracellular pathways might all act through a common mechanism involving the synthesis of ROI. ROI appear to serve as messengers mediating directly or indirectly the release of the inhibitory subunit I kappa B from NF-kappa B.

3,793 citations

Journal ArticleDOI
28 May 1998-Nature
TL;DR: The data suggest that two global mechanisms of gene regulation, DNA methylation and histone deacetylation, can be linked by MeCP2, an abundant nuclear protein that is essential for mouse embryogenesis.
Abstract: Cytosine residues in the sequence 5'CpG (cytosine-guanine) are often postsynthetically methylated in animal genomes. CpG methylation is involved in long-term silencing of certain genes during mammalian development and in repression of viral genomes. The methyl-CpG-binding proteins MeCP1 and MeCP2 interact specifically with methylated DNA and mediate transcriptional repression. Here we study the mechanism of repression by MeCP2, an abundant nuclear protein that is essential for mouse embryogenesis. MeCP2 binds tightly to chromosomes in a methylation-dependent manner. It contains a transcriptional-repression domain (TRD) that can function at a distance in vitro and in vivo. We show that a region of MeCP2 that localizes with the TRD associates with a corepressor complex containing the transcriptional repressor mSin3A and histone deacetylases. Transcriptional repression in vivo is relieved by the deacetylase inhibitor trichostatin A, indicating that deacetylation of histones (and/or of other proteins) is an essential component of this repression mechanism. The data suggest that two global mechanisms of gene regulation, DNA methylation and histone deacetylation, can be linked by MeCP2.

3,396 citations


Cites methods from "Accurate transcription initiation b..."

  • ...The coated beads were then incubated with 60 μg HeLa cell nuclear extrac...

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Journal ArticleDOI
13 Nov 1992-Cell
TL;DR: Three participants are identified (AT gene(s), p53, and GADD45) in a signal transduction pathway that controls cell cycle arrest following DNA damage; abnormalities in this pathway probably contribute to tumor development.

3,098 citations


Cites methods from "Accurate transcription initiation b..."

  • ...Gel Mobility Shift Assay Nuclear extracts were prepared as described proviously (Dignam et al., 1983; Carrier et al., 1992)....

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Journal ArticleDOI
TL;DR: A functionally tripartite, 50-nt hypoxia-inducible enhancer which binds several nuclear factors, one of which is induced by Hypoxia via de novo protein synthesis.
Abstract: We have identified a 50-nucleotide enhancer from the human erythropoietin gene 3'-flanking sequence which can mediate a sevenfold transcriptional induction in response to hypoxia when cloned 3' to a simian virus 40 promoter-chloramphenicol acetyltransferase reporter gene and transiently expressed in Hep3B cells Nucleotides (nt) 1 to 33 of this sequence mediate sevenfold induction of reporter gene expression when present in two tandem copies compared with threefold induction when present in a single copy, suggesting that nt 34 to 50 bind a factor which amplifies the induction signal DNase I footprinting demonstrated binding of a constitutive nuclear factor to nt 26 to 48 Mutagenesis studies revealed that nt 4 to 12 and 19 to 23 are essential for induction, as substitutions at either site eliminated hypoxia-induced expression Electrophoretic mobility shift assays identified a nuclear factor which bound to a probe spanning nt 1 to 18 but not to a probe containing a mutation which eliminated enhancer function Factor binding was induced by hypoxia, and its induction was sensitive to cycloheximide treatment We have thus defined a functionally tripartite, 50-nt hypoxia-inducible enhancer which binds several nuclear factors, one of which is induced by hypoxia via de novo protein synthesis

2,523 citations

Journal ArticleDOI
29 Aug 1986-Cell
TL;DR: In this paper, an electrophoretic mobility shift assay with end-labeled DNA fragments was used to characterize proteins that bind to the immunoglobulin (Ig) heavy chain and the kappa light chain enhancers.

2,413 citations

References
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Journal ArticleDOI
19 Sep 1980-Science
TL;DR: Sequences which are essential for the initiation of specific transcription in vitro were shown to be located between 12 and 32 base pairs upstream from the 5' end of these genes.
Abstract: In vitro genetic techniques were used to study the sequence requirements for the initiation of specific transcription. Deletion mutants were constructed around the putative promoter of the adenovirus-2 major late and chicken conalbumin genes. Specific transcription in vitro by RNA polymerase B together with a HeLa cell cytoplasmic extract was used as the test for promoter function. With this approach sequences which are essential for the initiation of specific transcription in vitro, were shown to be located between 12 and 32 base pairs upstream from the 59 end of these genes.

817 citations

Journal ArticleDOI
01 Oct 1979-Cell
TL;DR: Specific transcription initiation at the late promoter is observed with extracts derived from either virus-infected or uninfected KB cells and with class II RNA polymerases isolated from either human calf, murine or amphibian cells.

584 citations

Journal ArticleDOI
TL;DR: A soluble extract prepared from human cells (KB S-100) has been recently shown to direct accurate transcription initiation by purified RNA polymerase II at the major late promoter of adenovirus 2, and four components that are required for the active and selective initiation of transcription by RNA polymerases II at this promoter are identified.

488 citations

Journal ArticleDOI
TL;DR: It is argued that the T-A-T-A -A-A and A-G-A motif is a specificity element, a selector of eukaryotic gene transcription, and that deletion of H2A gene-specific conserved DNA sequences upstream from this motif enhanced mRNA synthesis.
Abstract: Conserved DNA sequence elements of putative regulatory functions were deleted from the prelude region of a sea urchin H2A histone gene. For this, the wild-type H2A gene of the 6-kilobase histone DNA repeat unit was replaced by various mutant H2A genes by cloning. The effects of the manipulation on H2A mRNA synthesis were studied by injection of the mutant DNAs into centrifuged Xenopus oocytes. The unmanipulated H2B gene residing within the same repeat unit provided a suitable internal control for these studies. Deletion of the T-A-T-A-A-A-T-A motif, once thought to be the functional equivalent of the bacterial Pribnow box, did not abolish transcription of the gene; instead, a number of novel mRNA 5' termini were generated. We argue that the T-A-T-A-A-A-T-A motif is a specificity element, a selector of eukaryotic gene transcription. Deletion of the "cap-sequence," 5' pyrimidine-C-A-T-T-C-purine 3' and most of the mRNA leader sequence did not abolish transcription but created yet another mRNA 5' terminus. In contrast to these deletions, which are both down-mutations, deletion of H2A gene-specific conserved DNA sequences upstream from the T-A-T-A-A-A-T-A motif enhanced mRNA synthesis. A hypothesis for the function of these DNA sequences as eukaryotic promoter elements is discussed.

338 citations

Journal ArticleDOI
TL;DR: Fractionated the KB S-100 and have found that multiple components are essential for the accurate transcription of these genes.

308 citations