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At least 253 records · Page 14Linked to original sources
PubMed1995

RNA polymerase II C-terminal domain required for enhancer-driven transcription.

The RNA polymerase II carboxy-terminal domain (CTD) consists of tandem repeats of the sequence Tyr-Ser-Pro-Thr-Ser-Pro-Ser. The CTD may participate in activated transcription through interaction with a high-molecular-weight mediator complex. Such a role would be consistent with observations that some genes are preferentially sensitive to CTD mutations. Here we investigate the function of the mouse RNA polymerase CTD in enhancer-driven transcription. Transcription by alpha-amanitin-resistant CTD-deletion mutants was tested by transient transfection of tissue culture cells in the presence of alpha-amanitin in order to inhibit endogenous RNA polymerase II. Removal of most of the CTD abolishes transcriptional activation by all enhancers tested, whereas transcription from promoters driven by Sp1, a factor that typically activates housekeeping genes from positions proximal to the initiation sites, is not affected. These findings show that the CTD is essential in mediating 'enhancer'-type activation of mammalian transcription.

Amanitins↗
PubMed2004

In situ imaging and isolation of proteins using dsDNA oligonucleotides.

As proteomics initiatives mature, the need will arise for the multiple visualization of proteins and supramolecular complexes within their true context, in situ. Single-stranded DNA and RNA aptamers can be used for low resolution imaging of cellular receptors and cytoplasmic proteins by light microscopy (LM). These techniques, however, cannot be applied to the imaging of nuclear antigens as these single-stranded aptamers bind endogenous RNA and DNA with high affinity. To overcome this problem, we have developed a novel method for the in situ detection of proteins using double-stranded DNA oligonucleotides. To demonstrate this system we have utilized the prokaryotic DNA-binding proteins LacI and TetR as peptide tags to image fusion proteins in situ using dsDNA oligonucleotides encoding either the Lac or Tet operator. Using fluorescent and fluorogold dsDNA oligonucleotides, we localized within the nucleus a TetR-PML fusion protein within promyelocytic leukaemia protein (PML) bodies by LM and a LacI-SC35 fusion protein within nuclear speckles by correlative light and electron microscopy (LM/EM). Isolation of LacI-SC35 was also accomplished by using biotinylated dsDNA and streptavidin sepharose. The use of dsDNA oligonucleotides should complement existing aptamer in situ detection techniques by allowing the multiple detection and localization of nuclear proteins in situ and at high resolution.

Animals↗
PubMedDate not supplied

[Activity of polynucleotide phosphorylase in ribosomal fraction of rat liver].

A method of isolating polynucleotidephosphorylase (PNPase) containing polyribosome fraction from rat liver is described. PNPase is found to be bind to RNA in polyribosomes with weak electrostatic bonds which are easily broken down in a weak alkaline medium with ionic strength more than 0.1 beta-22P-labelled ADP, GDP, UDP and CDP are found among the products of endogenous RNA degradation in the fraction of total polyribosomes in the presence of 32P-orthophosphate. A considerable change in the base composition of PNP-degraded RNA is observed at different incubation times of total polyribosomes with 32P-orthophosphate: G+C//A+U ratio increased from 2.3 to 3.1, and purines/pyrimidines ratio-from 0.47 to 1.06 with the increase of the incubation time. Specific activity of PNP in ribosome fractions obtained under ultracentrifugation of total polyribosomes in succrose density gradient (0.3-1.0 M) increased in the direction from the fraction of heavy polysomes to trimers and dimers and then dropped at the region of monomers (80 S particles). The data obtained give no possibility to determine the type of PNP-bound RNA in polyribomes of rat liver.

Animals↗
PubMed2003

Limitations on geminivirus genome size imposed by plasmodesmata and virus-encoded movement protein: insights into DNA trafficking.

Animals and plants evolved systems to permit non-cell-autonomous trafficking of RNA, whereas DNA plays a cell-autonomous role. In plants, plasmodesmata serve as the conduit for this phenomenon, and viruses have evolved to use this pathway for the spread of infectious nucleic acids. In this study, a plant DNA virus was used to explore the constraints imposed on the movement of DNA through this endogenous RNA trafficking pathway. The combined properties of the geminivirus-encoded movement protein and plasmodesmata were shown to impose a strict limitation on the size of the viral genome at the level of cell-to-cell movement. Size-increased viral genome components underwent homologous and nonhomologous recombination to overcome this strict limitation. Our results provide insights into the genetic mechanisms that underlie viral evolution and provide a likely explanation for why relatively few types of plant DNA viruses have evolved: they would have had to overcome the constraints imposed by an endogenous system operating to ensure that DNA acts in a cell-autonomous manner.

Cloning, Molecular↗
PubMed1981

Activation of the major drosophila heat-shock genes in vitro.

Inactive Drosophila heat-shock genes of isolated diploid nuclei can be induced to a transcriptionally active state by exposure to cytoplasmic extracts from heat-shocked Drosophila cells. No effect was observed on histone gene transcription, and extracts from non-heat-shocked cells were ineffective. The factor in the cytoplasmic extract has been partially purified and characterized. It is protease-sensitive and heat-labile. A striking change accompanies in vitro activation that permits transcription by E. coli RNA polymerase of the chromatin 5' -distal to the structural genes at the 87A and 87C heat-shock gene loci; we have previously observed a similar change after in vivo heat-shock induction. That this change occurred in the absence of endogenous RNA polymerase II activity suggests that these changes may reflect the earliest event in gene activation. Inasmuch as activation also took place after histone H1 depletion, this histone does not appear to be essential for this step of gene activation.

Animals↗
PubMedDate not supplied

Study of the type of RNA, degraded by polynucleotide phosphorylase in polyribosomal fraction of rat liver.

The type of RNA is studied, which is degraded by polynucleotide phosphorylase (PNPase) in the fraction of free ribosomes and ribosomes released from endoplasmic reticulum membranes with Triton X-100. Beta-32P labelled ADP, UDP, GDP and CDP are found among the degradation products of endogenous RNA of free and bound ribosomes in vitro in the presence of 32P-ortophosphate. An analysis of molar ratio of beta-32P-NDP isolated revealed that PNPase degrades RNA of GC type in both ribosome fractions. The amount of PNPase-degraded RNA in bound ribosimes is 4-fold as high as that in free ribosomes under the same conditions. Analysis of stable 32P-RNA and rapidly labelled 32-P-dRNA, isolated from bound ribosomes after the incubation with and without inorganic phosphate, revealed that PNPase attacks the 28S fragment of RNA, which consists of about 370 nucleotides, and dRNA having a sedimentation coefficient less than 12S. The rate of dRNA degradation is considerably higher than that of rRNA. 5'-RNAase, hydrolysing synthetic homopolyribonucleotides to oligonucleotides with free 3'-OH terminal group, apparently participates, together with PNPase, in dRNA and rRNA degradation.

Animals↗
PubMed1986

Biochemical and immunological characterization of Suncus murinus mammary tumor virus DNA polymerase.

Enzymological properties of the RNA-directed DNA polymerase associated with the Suncus murinus mammary tumor virus (Sm-MTV) was investigated and its antigenic relatedness to other retroviral DNA polymerases was examined. The enzyme exhibited higher activity in the presence of Mg2+ than in the presence of Mn2+ with endogenous RNA as well as with almost all of the synthetic template X primers tested. Mg2+ was also effective with poly(2'-O-methylcytidylate) X oligodeoxyguanylate which was known to be specific for Mn2+. To examine the immunological relatedness of this enzyme with other retroviral DNA polymerases, remaining Sm-MTV DNA polymerase activity was measured after treatment of this enzyme with various antisera prepared against each of the reverse transcriptases of Mason-Pfizer monkey virus (MPMV), murine mammary tumor virus (MuMTV), simian sarcoma virus-simian sarcoma associated virus (SSV/SSAV), and Rauscher murine leukemia virus (RLV). No inhibition of the Sm-MTV enzyme activity was observed when treated with the latter three antisera with which the DNA polymerase activities of the corresponding retroviruses were fully inhibited. Only the antiserum against MPMV-enzyme, however, was found to slightly inhibit the Sm-MTV enzyme activity. These results indicate that Sm-MTV DNA polymerase has similar enzymological properties to those of MPMV and MuMTV and shares some common antigenic determinant group(s) with MPMV DNA polymerase.

Animals↗
PubMed1983

Reconstitution of nucleoprotein complexes with mammalian heterogeneous nuclear ribonucleoprotein (hnRNP) core proteins.

Newly transcribed heterogeneous nuclear RNA (hnRNA) in the eucaryote cell nucleus is bound by proteins, giving rise to large ribonucleoprotein (RNP) fibrils with an inherent substructure consisting largely of relatively homogeneous approximately 20-nm 30S particles, which contain core polypeptides of 34,000-38,000 mol wt. To determine whether this group of proteins was sufficient for the assembly of the native beaded nucleoprotein structure, we dissociated 30S hnRNP purified from mouse ascites cells into their component proteins and RNA by treatment with the ionic detergent sodium deoxycholate and then reconstituted this complex by addition of Triton X-100 to sequester the deoxycholate. Dissociation and reassembly were assayed by sucrose gradient centrifugation, monitoring UV absorbance, protein composition, and radiolabeled nucleic acid, and by electron microscopy. Endogenous RNA was digested and reassembly of RNP complexes carried out with equivalent amounts of exogenous RNA or single-stranded DNA. These complexes are composed exclusively of groups of n 30S subunits, as determined by sucrose gradient and electron microscope analysis, where n is the length of the added nucleic acid divided by the length of nucleic acid bound by one native 30S complex (about 1,000 nucleotides). When the nucleic acid: protein stoichiometry in the reconstitution mixture was varied, only complexes composed of 30S subunits were formed; excess protein or nucleic acid remained unbound. These results strongly suggest that core proteins determine the basic structural properties of 30S subunits and hence of hnRNP. In vitro construction of RNP complexes using model nucleic acid molecules should prove useful to the further study of the processing of mRNA.

Animals↗
PubMed1981

Stimulatory effect of hnRNA on template activity of isolated rat liver chromatin.

Transcription of chromatin isolated from rat liver with E. coli RNA polymerase was stimulated up to five-fold with heterogeneous nuclear RNA (hnRNA) from rat liver. The transcription product had features of DNA-primed RNA. Neither cytoplasmic poly(A)-containing RNA nor high or low molecular weight cytoplasmic poly(A)-lacking RNA from rat liver exhibited stimulation of transcription. The stimulatory effect seems to be confined to middle repetitive sequences of hnRNA. With purified DNA templates, addition of hnRNA resulted in complete inhibition of transcription. The stimulatory effect of hnRNA on chromatin transcription was also observed with endogenous RNA polymerase. This effect was dependent ionic strength and was most pronounced under conditions optimal for RNA-polymerase B activity.

Ammonium Sulfate↗
PubMed1978

Mapping of adenovirus late promoters with nascent mercurated RNA.

Nascent RNA molecules were labeled in vivo and elongated in vitro by incubation of the isolated nuclei in the presence of mercurated nucleotides. The RNA molecules initiated and labeled in vivo and elongated in vitro were then selectively purified on a thiopropyl 6-B Sepharose affinity column. The procedure was shown to be free of artifacts since the addition of mercurated nucleotides and the retention on the affinity column is mediated by the endogenous RNA polymerase II (nucleoside triphosphate:RNA nucleotidyltransferase; EC 2.7.7.6), is sensitive to actinomycin D, and is dependent on the presence of all four ribonucleotide triphosphates. This general procedure was applied to the mapping of viral promoters late after adenovirus 2 infection of HeLa cells. RNA purified as described above was hybridized to restriction enzyme fragments attached to nitrocellulose filters. The 5' ends of the nascent RNA chains are located in coordinates 9.5-17 for a rightward transcript, 0-25 for a leftward transcript, and possibly 60-70 for a second rightward transcript. These locations clearly differ from locations of the early promoters and therefore suggest that the transition from early to late functions is controlled at the transcriptional level.

Adenoviruses, Human↗
PubMed1996

Two transcription factors related with the eucaryal transcription factors TATA-binding protein and transcription factor IIB direct promoter recognition by an archaeal RNA polymerase.

We reported previously that cell-free transcription in the Archaea Methanococcus and Pyrococcus depends upon two archaeal transcription factors, archaeal transcription factor A (aTFA) and archaeal transcription factor B (aTFB). In the genome of Pyrococcus genes encoding putative homologues of eucaryal transcription factors TATA-binding protein (TBP) and TFIIB have been detected. Here, we report that Escherichia coli synthesized Pyrococcus homologues of TBP and TFIIB are able to replace endogenous aTFB and aTFA in cell-free transcription reactions. Antibodies raised against archaeal TBP and TFIIB bind to polypeptides of identical molecular mass in the aTFB and aTFA fraction. These data identify aTFB as archaeal TBP and aTFA as the archaeal homologue of TFIIB. At the Pyrococcus glutamate dehydrogenase (gdh) promoter these two bacterially produced transcription factors and endogenous RNA polymerase are sufficient to direct accurate and active initiation of transcription. DNase I protection experiments revealed Pyrococcus-TBP producing a characteristic footprint between position -20 and -34 centered around the TATA box of gdh promoter. Pyrococcus-TFIIB did not bind to the TATA box but bound cooperatively with Pyrococcus-TBP generating an extended DNase I footprinting pattern ranging from position -19 to -42. These data suggest that the Pyrococcus homologue of TFIIB associates with the TBP-promoter binary complex as its eucaryal counterpart, but in contrast to eucaryal TFIIB, it causes an extension of the protection to the region upstream of the TATA box.

Archaea↗
PubMed1996

An RNA-export mediator with an essential nuclear export signal.

The Rev protein of human immunodeficiency virus type 1 (HIV-1) mediates the translocation of viral messenger RNAs from the nucleus to the cytoplasm. In yeast, Rev can mediate the nuclear export of Rev response-element-containing RNAs. The export of Rev itself proceeds through the nuclear pore complex and requires a nuclear export signal (NES) and interaction with a cellular cofactor, the protein Rip1. Endogenous RNA export mediators that interact with Rip1 and harbour NESs are thought to exist but have yet to be identified. Here we report the characterization of a new and essential yeast protein, Gle1, which contains an NES and has a relative molecular mass of 62,000. Mutation of the NES in Gle1 prevents export of polyadenylated RNA from the nucleus. Gle1 interacts with Rip1 and the nucleoporin Nup100 and is localized predominantly at nuclear pore complexes. These properties indicate that Gle1 is an RNA-export factor and that Rev may mediate viral RNA export by mimicking the function of Gle1.

Amino Acid Sequence↗
PubMed1977

Evolutionary relationships between gag gene-coded proteins of murine and primate endogenous type C RNA viruses.

Several low molecular weight proteins of endogenous type C viruses of the RD114/baboon group are compared with the gag gene translational products of endogenous type C viruses of murine origin. The p10 proteins of each virus group are shown to be immunologically and biochemically related, while the p12 proteins of RD114/baboon viruses are demonstrated to share antigenic determinants with murine viral p15. Moreover, highly type-specific phosphoproteins, p15 of RD114/baboon viruses and p12 of murine viruses, are shown to possess very similar biochemical properties. These findings, along with previous studies indicating immunologic cross-reactivity between their major internal antigens, p30, demonstrate that each of the gag gene-coded proteins of murine type C viruses has a analogue in viruses of the RD114/baboon group. The immunologic and biochemical relatedness of their gag gene translational products supports the concept of a common progenitor in the evolution of these endogenous viruses.

Animals↗
PubMed1999

Human Nopp140, which interacts with RNA polymerase I: implications for rRNA gene transcription and nucleolar structural organization.

Nopp140 is thought to shuttle between nucleolus and cytoplasm. However, the predominant nucleolar localization of Nopp140 homologues from different species suggests that Nopp140 is also involved in events occurring within the nucleolus. In this study, we demonstrated that the largest subunit of RNA polymerase I, RPA194, was coimmunoprecipitated with the human Nopp140 (hNopp140). Such an interaction is mediated through amino acids 204 to 382 of hNopp140. By double immunofluorescence, hNopp140 was colocalized with RNA polymerase I at the rDNA (rRNA genes) transcription active foci in the nucleolus. These results suggest that Nopp140 can interact with RNA polymerase I in vivo. Transfected cells expressing the amino-terminal half of hNopp140, hNopp140N382 (amino acids 1 to 382), displayed altered nucleoli with crescent-shaped structures. This phenotype is reminiscent of the segregated nucleoli induced by actinomycin D treatment, which is known to inhibit rRNA synthesis. Consistently, the hNopp140N382 protein mislocalized the endogenous RNA polymerase I and shut off cellular rRNA gene transcription as revealed by an in situ run-on assay. These dominant negative effects of the mutant hNopp140N382 suggest that Nopp140 plays an essential role in rDNA transcription. Interestingly, ectopic expression of hNopp140 to a very high level caused the formation of a transcriptionally inactive spherical structure occupying the entire nucleolar area which trapped the RNA polymerase I, fibrillarin, and hNopp140 but excluded the nucleolin. The mislocalizations of these nucleolar proteins after hNopp140 overexpression imply that Nopp140 may also play roles in maintenance of nucleolar integrity.

Animals↗
PubMed1990

Functional acetylcholine receptors expressed in Xenopus oocytes after injection of Torpedo beta, gamma, and delta subunit RNAs are a consequence of endogenous oocyte gene expression.

The nicotinic acetylcholine receptor (AChR) is an oligomeric transmembrane glycoprotein consisting of four homologous subunits in a stoichiometry alpha 2 beta gamma delta. Xenopus oocytes were used to study the effects of selectively deleting the alpha subunit of the Torpedo californica AChR on functional receptor expression. Oocytes microinjected with only Torpedo beta, gamma, and delta subunit RNAs showed small acetylcholine-elicited currents. These "alpha-less" AChRs were pharmacologically similar to the wild-type (i.e., Torpedo alpha 2 beta gamma delta) receptor. Actinomycin D, which blocks endogenous RNA transcription, completely inhibited the expression of alpha-less but not wild-type receptor. Coinjection of antisense RNA to the alpha subunit of the Xenopus muscle AChR with Torpedo beta, gamma, and delta subunit RNAs significantly reduced expression of the alpha-less AChRs without altering expression of wild-type receptors. These results indicate that Xenopus oocytes express low levels of AChR alpha subunit mRNA that, when translated, can lead to the formation of functional Xenopus-Torpedo AChR hybrids. These results strongly suggest that, unless the potential contribution of endogenous subunits can be determined, caution must be exercised when analyzing the effects of subunit deletions on multisubunit protein expression in Xenopus oocytes.

Acetylcholine↗
PubMed1995

Distribution of RNase MRP RNA during Xenopus laevis oogenesis.

RNase MRP is a ribonucleoprotein endoribonuclease found predominantly in nucleoli, but which has been associated with mitochondria and mitochondrial RNA processing. In order to analyze the intracellular localization of specific RNA components of ribonucleoproteins of this type, a whole-mount method for in situ hybridization in Xenopus laevis oocytes was employed. Results with specific probes (for both mitochondrial and nonmitochondrial RNAs) indicate that this procedure is generally effective for the detection of a variety of nucleic acids that reside in different cellular compartments. Probes used to detect the endogenous RNA component of RNase MRP (MRP RNA) during X. laevis oogenesis revealed a continuous nuclear signal as well as a possible dual localization of MRP RNA in nucleoli and mitochondria at developmental stages temporally consistent with both ribosomal and mitochondrial biogenesis. Genomic DNA encoding MRP RNA was injected into the nuclei of stage VI oocytes and correctly transcribed. The in vivo-transcribed RNA was properly assembled with at least some of its cognate proteins as demonstrated by immunoprecipitation with specific autoantiserum. In addition, detectable levels of the RNA were exported to the cytoplasm. This whole-mount procedure has permitted us to identify MRP RNA in situ at different developmental time points as well as during transcription of the injected gene, and suggests differential localization of MRP RNA during oogenesis consistent with its proposed function in both mitochondria and nucleoli.

Animals↗
PubMed1978

Folded chromosomes of vegetative Bacillus subtilis: composition and properties.

The isolation of folded DNA from Bacillus subtilis, a Gram positive bacterium is described. When the lysis was achieved with 1 M NaCl a slow sedimenting nucleoid was obtained (1600-2000 S). Conversely, when the lysis was achieved with 0.2 M NaCl a fast sedimenting nucleoid was obtained (3500-4000 S). The yield of folded DNA was between 80 to 90 % of the total lysate DNA. Both nucleoids contained the same amount of RNA, but the relative proportions of lipids and proteins were different. Folded chromosomes were prepared in the presence of spermidine: artifactual protein binding is shown to be unlikely. Electrophoresis of nucleoid proteins showed a dominant polypeptide (MW = 36,000), which remained associated with DNA after sarcosyl treatment and could be partially removed by heat mediated DNA unfolding. In vitro transcription by endogenous RNA polymerase bound to the fast sedimenting-nucleoid was rifamycin resistant; the template capacity of the fast sedimenting-nucleoid was compared with that of the completely unfolded chromosomes.

Bacillus subtilis↗
PubMed1983

Isolation of transcription factors that discriminate between different promoters recognized by RNA polymerase II.

A new fractionation procedure separates a whole-cell HeLa extract into three components required for accurate in vitro transcription. One component (Sp1) is a promoter-specific factor required for transcription of the SV40 early and late promoters, but not for transcription of other promoters we have tested. The second component (Sp2) is a general factor required for transcription of the SV40 promoters and a series of others, including the adeno-virus 2 major late promoter, the human beta-globin promoter and the avian sarcoma virus promoter. The third component is a fraction containing the endogenous RNA polymerase II. When SV40 and adenovirus templates were present simultaneously in an in vitro transcription reaction, addition of the Sp1 factor stimulated SV40 early promoter transcription 40-fold, and inhibited adenovirus major late promoter transcription by 40%. This finding suggests that Sp1 is involved in promoter selection, and is not merely a general transcription stimulatory factor.

DNA-Directed RNA Polymerases↗