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Biomedical subjects

E Wingender

Publications and source records attributed to E Wingender.

71 records · Page 4Linked to original sources
PubMed1988

Complete synthesis and transcription in vitro of a gene coding for human ribosomal 5S RNA.

The gene coding for the major human ribosomal 5S RNA was chemically synthesized and cloned into a pUC13 vector. This approach was taken, because attempts to isolate the human 5S gene have thus far yielded either pseudogenes or variant 5S genes of unknown function. The synthetic human gene was transcribed by RNA polymerase III either in a crude HeLa cell extract or in a system reconstituted from partially purified transcription factors. Comparative studies with the Xenopus laevis somatic 5S gene show that the human gene is transcribed with similar fidelity and an efficiency of about 80% under optimal conditions. The time-course of transcription and optimal concentrations of template and transcription factors were found to be similar for both genes studied. The synthetic gene described may prove useful to study its interaction with human transcription factors in a homologous system.

Animals↗
PubMed1987

Transcription complexes for various class III genes differ in parameters of formation and stability towards salt.

RNA polymerase III faithfully transcribes the genes for ribosomal 5 S RNA, tRNA(1Met) or adenovirus VA RNA in vitro in the presence of required transcription factors. These genes display distinct differences in the kinetics of transcription complex formation and in their response to excess template. In contrast to tRNA and VA RNA synthesis, 5 S RNA synthesis displays a lag phase of 15 minutes before the onset of transcription and is clearly inhibited by high concentrations of template. Once formed, transcription complexes for the RNA polymerase III genes listed can be isolated by glycerol gradient centrifugation and display a remarkable stability against transient treatment with high salt concentrations. Complexes for 5 S RNA and tRNA remain functionally active up to 2.5 M-KCl. The activity of transcription complexes for VA RNA, however, is significantly diminished after treatment with high salt concentrations. This effect is shown to be due to an irreversible loss of transcription factors. RNA polymerase III is dissociated by high salt concentrations from all the transcription complexes studied but remains part of these complexes during the normal reinitiation cycle at 60 mM-KCl. An additional method for the purification of partial transcription complexes was developed that involves equilibrium centrifugation on cesium sulfate gradients. This method completely releases TFIIIB from 5 S complexes and a core complex, composed of the 5 S RNA gene, factors IIIA and IIIC, is retained. In the case of tRNA and VA RNA, core complexes are obtained that remain partly associated with TFIIIC and TFIIIB. These results indicate a qualitatively and/or quantitatively different interaction of individual factors in different polymerase III transcription complexes.

Genes↗
PubMed1986

Association of RNA polymerase III with transcription factors in the absence of DNA.

The gene for tRNAMet1 from Xenopus oocytes was transcribed in a cell free system with components isolated from a HeLa cell-free extract. It was found that, apart from the established assembly of transcription factors IIIB and IIIC on tRNA genes into stable transcription complexes, these factors can also associate with the enzyme in the absence of DNA to form a functional polymerase III complex. These complexes can be isolated in a highly active form from the bulk of other cellular proteins by mild methods such as gel filtration or density gradient centrifugation. When associated with RNA polymerase III into a functional complex, the transcription factors IIIB and IIIC can clearly be differentiated from free transcription factors, which individually display a much lower relative molecular mass. The polymerase complexes are stable against 1 M KCl, rendering unlikely that they represent fortuitous aggregates including RNA polymerase III and transcription factors IIIB and IIIC. These complexes are sensitive to dilution and, whereas transcription factor IIIC binds to the enzyme more tightly, factor IIIB tends to leak from the complex upon dilution of the protein concentration. From these results it is clear that in addition to their function as DNA-binding protein(s), transcription factors IIIB and IIIC can directly interact with RNA polymerase III without prior binding to the promoter region of the gene to be transcribed.

Animals↗
PubMed1984

Zinc ions are differentially required for the transcription of ribosomal 5S RNA and tRNA in a HeLa-cell extract.

Chelation of divalent cations by 5 mM EDTA and subsequent removal by dialysis from a cytoplasmic HeLa cell extract leads to a complete loss of 5S rRNA transcription without affecting tRNA synthesis. Transcription complexes for 5S RNA can no longer be assembled in such a zinc-depleted extract and this ability can be fully restored only by the re-addition of 5 microM zinc. Reconstitution experiments with isolated protein fractions show that transcription factor A from HeLa-cells requires zinc to exert its specific function. Pre-formation of transcription complexes partially protects the metal ion against removal by chelation even in the presence of 1.8 M KCl. These results indicate that the zinc ions are bound to mammalian transcription factor IIIA which, in a transcription complex, binds very strongly to the 5S RNA gene. Cation depletion with 75 mM EDTA also suppresses tRNA transcription; an effect which is reversible by zinc addition. We conclude that beside for the binding of TF IIIA, zinc is also bound with a different affinity to a transcription component common to 5S and tRNA synthesis, most likely polymerase III itself.

Edetic Acid↗
PubMed1984

Isolation of a transcription complex for ribosomal 5S RNA.

Cloned 5S rRNA genes from Xenopus borealis oocytes can be used to assemble functional transcription complexes from cytoplasmic HeLa cell extracts as a source for polymerase III and all factors additionally required for faithful 5S RNA transcription. Such complexes can be isolated by glycerol gradient ultracentrifugation and non-denaturing gel electrophoresis. They contain less than 1% of the cellular protein and retain their fidelity to synthesize 5S rRNA. The assembly of the complex is unaffected by KCl concentrations up to 140 mM whereas the transcription of 5S rRNA by the isolated complex is significantly reduced at this ionic strength. This indicates that the latter process, involving re-initiation by RNA polymerase III, is more sensitive to elevated salt concentrations than is the assembly of the transcription complexes. Furthermore, we show that complex formation also takes place in the absence of exogenously added nucleoside triphosphates, although this results in a slight shift in the sedimentation position which can be reversed by addition of the initial nucleotides GTP and CTP. We have analyzed the isolated transcription complexes by the protein blotting technique in an attempt to characterize their DNA-binding components. The results show a single component, corresponding to a protein with a mol. wt. of approximately 45 kd, which binds selectively, but not exclusively to a DNA fragment containing the 5S gene. The possible relationship of this protein to transcription factor IIIA from Xenopus oocytes is discussed.

Animals↗
PubMed1983

Faithful transcription of ribosomal 5-S RNA in vitro depends on the presence of several factors.

Cytoplasmic extracts from HeLa cells, capable of transcribing the cloned genes for ribosomal 5-S RNA, were employed to study the factors involved in this process. Two factors can be isolated, by gel filtration through Sephadex G-100, which are devoid of RNA polymerase activity. They significantly enhance the extent and specificity of the transcription of 5-S rRNA. Both proteins can jointly be purified by affinity chromatography on immobilized DNA containing the genes for ribosomal 5-S RNA from Xenopus borealis. Besides a protein of approximately 45 kDa, possibly corresponding to TF IIIA isolated from Xenopus oocytes, a second protein with a molecular mass of 22 +/- 1 kDa stimulates the formation of 5-S RNA. This protein is contained in the breakthrough of DEAE-cellulose; it binds to and is eluted from phosphocellulose with 0.6 M KCl. In addition, it was found that the exclusion volume obtained after gel filtration on Sephadex G-100 contains functional complexes, which are capable of transcribing the cloned 5-S genes and hence contain all the required factors. Direct evidence is presented that the protein of 22 kDa described above is contained in and can be isolated from such complexes. It is postulated from indirect evidence that an additional factor with a molecular mass in excess of 100 kDa is required which can be removed from functional polymerase complexes by gel filtration through Bio-Gel A5m.

Chromatography, DEAE-Cellulose↗
PubMed1982

Parameters affecting the superreactivity of the cysteine side chain in histone H3. Characterization of a 32-amino-acid peptide including Cys-110.

Tryptic digestion of the whole histone mixture from chicken erythrocytes is used to isolate the hydrophobic portion (residues 84-115) of histone H3. The phenomenon of a superreactive thiol at Cys-110 was reinvestigated by the reaction of a neutral, fluorogenic reagent, N-[p-(2-benzimidazolyl)phenyl]maleimide, with this peptide and with the intact histone. Removing the highly basic portions of H3 leads to a strong reduction of Cys-110 reactivities, even if a stoichiometric complex with histone H4 is reconstituted. An apparent superreactivity is found only for a non-degraded histone H3 under conditions of low ionic strength but not for the histone complex in its native conformation. It is concluded that thiol activation is due to an artifactual cluster of positive charges around Cys-110 rather than to a stable microenvironment.

Amino Acid Sequence↗
PubMed1980

Modulation of the nucleosome structure by histone acetylation.

A rapid procedure for the isolation of core particles from Chinese hamster ovary cells is described which permits measurements, usually at the day of their preparation. Particles of 145 +/- 5 base pairs, derived from interphase cells, will be compared with the analogue specimens from butyrate-treated cells, metaphase cells and a standard preparation from chicken erythrocytes. Butyrate cause an increase in the acetylation of histones H3 and H4, which induces alterations of the interhistone and histone-DNA interactions. Changes in the interhistone contacts, correlated to an extension of alpha-helical segments, lead to an altered accessibility of the H3 cysteine side-chains and to a different histone displacement by protamines. On the other hand, histone-DNA contacts are loosened in parts and this is particularly evident from the changes in the premelting region of a thermal-denaturation profile.

Acetylation↗
PubMedDate not supplied

[Differentiation of osteogenetic cells: systems and regulators].

Bone formation comprises a complex but ordered sequence of events, beginning with the proliferation of chondrogenic and osteogenic precursor cells followed by their subsequent differentiation, ultimately leading to extracellular matrix maturation and mineralisation. Several models have been established which recreate discrete elements of this network. Factors induce ectopic bone formation, when implanted into muscle pouches, have been characterized as members of the TGF-beta-superfamily. Detailed information about the course of OB-differentiation has been obtained from an in vitro model system. The process of mineralisation was found to consist of three distinct time periods: a proliferative phase, a period of extracellular matrix maturation and mineralisation. The development of each states depends on each other. This model is not as complex as the whole organ and cannot of course lead to any conclusion about the kinetic differentiation path of a cell in its normal spatial environment. Different organ culture systems are described and through the application of sensitive methods the differentiation can be studied in the normal spatial environment. The cascade of events in the differentiation process must be strictly regulated. Hormones and growth factors in many cases show a bone-forming and/or bone-resorbing action. The effects of the classical calcium regulating hormones Vitamin D and PTH on OB-differentiation are reviewed. A large number of growth factors have been shown to effect OB. Growth factors, that have been isolated from the bone matrix are of particular interest to bone formation.

Animals↗
PubMedDate not supplied

Integrated databases and computer systems for studying eukaryotic gene expression.

MOTIVATION: The goal of the work was to develop a WWW-oriented computer system providing a maximal integration of informational and software resources on the regulation of gene expression and navigation through them. Rapid growth of the variety and volume of information accumulated in the databases on regulation of gene expression necessarily requires the development of computer systems for automated discovery of the knowledge that can be further used for analysis of regulatory genomic sequences. RESULTS: The GeneExpress system developed includes the following major informational and software modules: (1) Transcription Regulation (TRRD) module, which contains the databases on transcription regulatory regions of eukaryotic genes and TRRD Viewer for data visualization; (2) Site Activity Prediction (ACTIVITY), the module for analysis of functional site activity and its prediction; (3) Site Recognition module, which comprises (a) B-DNA-VIDEO system for detecting the conformational and physicochemical properties of DNA sites significant for their recognition, (b) Consensus and Weight Matrices (ConsFrec) and (c) Transcription Factor Binding Sites Recognition (TFBSR) systems for detecting conservative contextual regions of functional sites and their recognition; (4) Gene Networks (GeneNet), which contains an object-oriented database accumulating the data on gene networks and signal transduction pathways, and the Java-based Viewer for exploration and visualization of the GeneNet information; (5) mRNA Translation (Leader mRNA), designed to analyze structural and contextual properties of mRNA 5'-untranslated regions (5'-UTRs) and predict their translation efficiency; (6) other program modules designed to study the structure-function organization of regulatory genomic sequences and regulatory proteins. AVAILABILITY: GeneExpress is available at http://wwwmgs.bionet.nsc. ru/systems/GeneExpress/ and the links to the mirror site(s) can be found at http://wwwmgs.bionet.nsc.ru/mgs/links/mirrors.html+ ++.

Algorithms↗