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D Rungger

Publications and source records attributed to D Rungger.

18 recordsLinked to original sources
PubMed1992

The weak, fine-tuned binding of ubiquitous transcription factors to the Il-2 enhancer contributes to its T cell-restricted activity.

The T lymphocyte-specific enhancers of the murine and human Interleukin 2 (Il-2) genes harbour several binding sites for ubiquitous transcription factors. All these sites for the binding of AP-1, NF-kB or Oct-1 are non-canonical sites, i.e. they differ in one or a few base pairs from consensus sequences for the optimal binding of these factors. Although the factors bind weakly to these sites, the latter are functionally important because their mutation to non-binding sites results in a decrease of inducible activity of the Il-2 enhancer. Conversion of three sites to canonical binding sites of Octamer factors, AP-1 and NF-kB results in a drastic increase in enhancer activity and the induction of the Il-2 enhancer in non-T cells, such as B cell lines, murine L cells and human HeLa cells. The introduction of two or three canonical sites into the enhancer leads to a further increase of its activity. Il-2 enhancer induction is also observed in B cells when the concentration of AP-1 and Oct factors increases as a result of cotransfections with FosB and Octamer expression plasmids. When Il-2 enhancer constructs carrying canonical factor binding sites were injected into Xenopus oocytes the strong binding of ubiquitous factors substantially overcomes the silencing effect of negatively acting factors present in resting primary T lymphocytes. These results suggest a fine-tuned interplay between ubiquitous and lymphoid-specific factors binding to and transactivating the Il-2 enhancer and show that the binding affinity of ubiquitous factors to the enhancer contributes to its cell-type specific activity. Moreover, we believe that a dramatic increase of transcriptional activity brought about by single point mutations at strategic important factor binding sites may also have relevance to the activation of nuclear oncogenes.

Animals↗
PubMed1992

Drosophila acetylcholinesterase. Expression of a functional precursor in Xenopus oocytes.

In insects, acetylcholinesterase is mainly found in the central nervous system. It is expressed in the synapse where it hydrolyzes the neurotransmitter acetylcholine. Maturation of this protein involves several post-translational modifications. The precursor polypeptide is cut at three sites; the N-terminal signal peptide is removed, the C-terminal hydrophobic polypeptide is clipped off and replaced by a glycolipid anchor and the resulting peptide is cut into two polypeptides, corresponding to active subunits. Two of these active subunits are associated to form the final active glycosylated protein. We have expressed the protein via microinjection of an expression vector into Xenopus oocyte nuclei. When the complete cDNA is injected, the acetylcholinesterase formed is biochemically similar to the Drosophila-head acetylcholinesterase. However, the hydrophobic C-terminal peptide is not replaced by a glycolipid anchor. As a consequence, the enzyme is no longer externalized, the proteolytic cutting of the main peptide does not occur and a new polymerization form occurs. Although incompletely processed, this protein is enzymatically active. When a cDNA lacking the coding region of the C-terminal hydrophobic peptide is injected, the resulting acetylcholinesterase is hydrophilic, cleaved into two subunits and secreted into the incubation medium free of contaminants.

Acetylcholinesterase↗
PubMed1992

Cyclosporin A and FK506 prevent the derepression of the IL-2 gene in mitogen-induced primary T lymphocytes.

In resting primary T lymphocytes the interleukin 2 (IL-2) gene is silenced by a repressor binding to the Pud element spanning positions -292 to -264 upstream of the cap site. Upon T-cell activation, this silencer is displaced by a positive transcription factor (TF) and the gene is derepressed and transcribed. Cyclosporin A (CsA) and FK506 interfere with normal derepression of the IL-2 gene. Both drugs exert no direct effect on basal transcription of the IL-2 or control viral genes. Direct addition does not abolish the active state of positive TFs present in proteins from activated T cells. However, if T cells are activated in the presence of either drug, their proteins not only fail to derepress, but efficiently and irreversibly silence IL-2 transcription. DNA-protein binding data show that proteins present in drug-treated cells form retarded complexes corresponding in size to the silencer and positive TF. Thus, in drug-treated cells a functional silencer persists, and a positive TF-like factor appears which is functionally abnormal. Moreover, drug-treated T cells appear to form a component that prevents functioning of normal positive TF.

Animals↗
PubMed1991

Silencing and trans-activation of the mouse IL-2 gene in Xenopus oocytes by proteins from resting and mitogen-induced primary T-lymphocytes.

The Xenopus oocyte system was used to test functionally, putative trans-active elements involved in the transcriptional control of the mouse interleukin-2 (IL-2) gene in resting and mitogen-induced primary T-lymphocytes. The IL-2 gene injected into the oocyte is active over a wide range of DNA concentrations. This basal activity is silenced by the addition of protein extracts from G0-arrested spleen cells. Extracts from 8 h-stimulated spleen cells do not silence but moderately increase transcription over basal level. When IL-2 transcription is silenced first by an injection of extract from resting spleen cells, the addition of proteins from stimulated cells results in a strong increase in transcription (derepression). Use of proteins from purified splenic T-lymphocytes shows that both silencer(s) and activator(s) are contributed by these cells. Extracts from control tissues have neither a silencing nor stimulatory effect. None of the proteins tested affects the activities of co-injected control genes. Injections with IL-2 promoter mutants indicate that the main target sequence of the silencing and activating factors is a purine region (Pu-box) lying between positions -261 and -292 upstream of the IL-2 gene. Bandshift assays show differential binding of the Pu-box with proteins from resting or activated T-cells.

Animals↗
PubMed1991

Neuronal nicotinic acetylcholine receptors expressed in Xenopus oocytes: role of the alpha subunit in agonist sensitivity and desensitization.

Neuronal nicotinic acetylcholine receptors (nAChRs) were expressed in Xenopus laevis oocytes after nuclear injection of complementary deoxyribonucleic acid (cDNA) expression vectors. The two receptor subtypes alpha 4/n alpha 1 and alpha 3/n alpha 1 were readily distinguishable from one another by ACh sensitivity and desensitization. alpha 3/n alpha 1 receptors showed lower ACh sensitivity and stronger desensitization than alpha 4/n alpha 1 receptors. Furthermore, although the current/voltage relationship was very similar in both receptor subtypes, the voltage dependence of desensitization was found to be strikingly different. As the n alpha 1 subunit was unchanged, the alpha subunits must be responsible for these functional differences. Symmetric hybrid alpha cDNAs, alpha 4:alpha 3 and alpha 3:alpha 4, were constructed and functional receptors were obtained by co-injection with n alpha 1. These hybrid receptors displayed an ACh sensitivity that was mainly defined by the extracellular sequence of the alpha subunit. In contrast, no part of the alpha subunit was found fully to determine desensitization.

Acetylcholine↗
PubMed1991

Steroids inhibit nicotinic acetylcholine receptors.

Application of progesterone to Xenopus oocytes expressing a cloned neuronal nicotinic acetylcholine (nAChR) revealed two effects. The first effect was a fully reversible reduction of the current induced by acetylcholine (ACh), its onset being nearly instantaneous. The second effect, which developed in a few hours, was an irreversible suppression of ACh-evoked currents. The transient inhibition had an apparent Ki of 7 microM when tested with 50 nM ACh, but the percentage of inhibition was positively correlated to the ACh concentration. A reduction of ACh-induced currents which appeared immediately upon progesterone application was also observed with muscle nAChR expressed in oocytes and with nAChR on membrane patches isolated from ciliary ganglion neurons. Thus nAChRs are modulated by progesterone and steroids may play an important role in nicotinic cholinoception.

Animals↗
PubMed1990

Alpha 5, alpha 3, and non-alpha 3. Three clustered avian genes encoding neuronal nicotinic acetylcholine receptor-related subunits.

In vertebrates, neuronal nicotinic acetylcholine receptors (nAChRs) assemble in an unknown stoichiometry from two homologous subunits, an alpha and a non-alpha. How large is the repertoire of these subunits and how many subtypes of functionally different nAChRs can they constitute? We found in the avian genome a cluster of three closely linked genes spanning 28 kilobase pairs and encoding three proteins, n alpha 3, alpha 3, and alpha 5, that have the features expected of neuronal nAChR subunits. Gene n alpha 3 lies 5' of alpha 3 (whose role in cholinoception has already been established) and is transcribed from the same DNA strand, whereas alpha 5 lies 3' of alpha 3 and is transcribed from the opposite DNA strand. The structure of the n alpha 3 and alpha 5 genes consists of six exons with precisely conserved splice sites and is identical to the structure of the previously characterized avian neuronal receptor subunit genes alpha 2, alpha 3, alpha 4, and n alpha 1. alpha 3, n alpha 3, and alpha 5 transcripts are rare in the central nervous system, but alpha 3 and n alpha 3 are readily detectable in embryonic superior cervical and ciliary ganglia. In order to assay function, the gene encoding n alpha 3 and the cDNAs encoding alpha 3, alpha 4, alpha 5, and n alpha 1 were subcloned into an expression vector, and the constructs were injected into Xenopus oocyte nuclei, either singly or in pairwise combinations of one alpha and one non-alpha. One to five days later, ACh sensitivity of the injected oocytes was examined in voltage clamp. The n alpha 3 gene and n alpha 1 cDNA elicited assembly of nAChRs when coinjected with alpha 3 or alpha 4 cDNA and the electrophysiological properties of the four pairwise combinations were significantly different. alpha 5, however, did not direct the assembly of functional nAChRs when injected alone or in combination with n alpha 1 or n alpha 3.

Acetylcholine↗
PubMed1990

Activation and blocking of neuronal nicotinic acetylcholine receptor reconstituted in Xenopus oocytes.

Neuronal nicotinic acetylcholine receptor of the alpha 4/non-alpha (alpha 4/n alpha) type was reconstituted in Xenopus oocytes after nuclear injection of cDNA expression vectors. Functional neuronal receptor was only formed when the two subunits alpha 4 and n alpha were coinjected, neither alpha 4 nor n alpha alone being effective. Responses to bath application of acetylcholine (AcCho) have been measured in voltage clamp. AcCho doses as low as 10 nM induce currents of up to 50 nA. Dose-response studies indicate a Kd of about 0.77 x 10(-6) M and a Hill coefficient of 1.5, thus predicting more than one AcCho binding site per receptor molecule. The current-voltage relationship of AcCho-induced currents presents a strong inward rectification. Responses to AcCho were compared to those of three other agonists: L-nicotine, carbachol, and 1,1-dimethyl-4-phenylpiperazinium iodide (DMPP). Sensitivities to AcCho, nicotine, and DMPP are quite similar. Sensitivity to carbachol is much lower, but the currents are otherwise indistinguishable from those induced by AcCho. Five AcCho antagonists--neuronal bungarotoxin (kappa-bungarotoxin), tubocurarine (TC), hexamethonium bromide (Hex), decamethonium bromide (Dec), and mecamylamine (Mec)--have been tested. Neuronal bungarotoxin has no effect on the alpha 4/n alpha channel, whereas 2.5 microM TC reduces by half the current peak evoked by 1 microM AcCho. The block by TC is independent of membrane voltage. By contrast, the block of AcCho-induced currents by Hex or Dec is strongly voltage dependent, suggesting that these substances enter the channel. The block by Mec is detectable at concentrations as low as 100 nM when applied together with 1 microM AcCho and is voltage independent. Hex, Dec, and Mec are effective only when AcCho is present. While the effects of all other agents are fully reversible, the Mec block is persistent.

Acetylcholine↗
PubMed1979

Intranuclear injection of anti-actin antibodies into Xenopus oocytes blocks chromosome condensation.

The role of contractile proteins in the structural organisation of the interphase nucleus and of metaphase chromosomes is largely unknown. Actin has been found in interphase nuclei of different species, especially in association with condensed chromatin. In the germinal vesicle (nucleus) of Xenopus oocytes, actin has been localised in the nuclear gel supporting the chromosomes and the extrachromosomal nucleoli. It has been reported that the premeiotic lampbrush chromosomes in these germinal vesicles are positively stained for actin and tubulin by the immunoperoxidase technique. Moreover, the longitudinal contraction of these chromosomes is ATP dependent. Therefore it has been suggested that actin participates in the structural organisation of the highly specialised lampbrush chromosomes. However, actin is not a major component of the metaphase chromosome scaffold. The results reported here suggest that actin is involved in the condensation of Xenopus chromosomes.

Actins↗
PubMed1979

The in vitro of Rous sarcoma virus RNA and function of the viral protein during the viral replication.

The gag gene and pol gene of the Rous sarcoma virus are translated in vitro from the 35S viral RNA. The env gene cannot be translated in vitro from the 35S RNA. For the in vitro translation of the src gene. 3' end fragments of the viral RNA are used. The gag protein p15 has a proteolytic activity and specifically processes its own protein precursor pr76. The gag protein p19 suppresses the in vitro translation of the pol gene.

Animals↗
PubMed1979

SV40 DNA injected into Xenopus oocyte nuclei is transcribed by RNA polymerase B.

SV40 DNA I. injected into Xenopus oocyte nuclei is transcribed. The SV40-specific RNA molecules migrate on sucrose gradients as do viral RNAs formed in infected green monkey cells but a variable proportion of RNA sequences complementary to SV40 DNA is also found in the light region of the gradients. All SV40-specific RNA species seem to be synthesized by RNA polymerase B as their synthesis is completely sensitive to low concentrations (0.1 microgram/ml) of alpha-amanitin. Concomittantly, the formation of SV40-specific proteins (tumor antigens) is inhibited by injecting alpha-amanitin together with the SV40 DNA.

Amanitins↗
PubMed1979

Transcription of spacer sequences in genes coding for ribosomal RNA in Xenopus cells.

Untreated Xenopus cells synthesize RNA is increased and some sequences complementary to those of the nontranscribed spacer are found in the heavy shoulder of the 40S precursor rRNA peak. In such events transcription initiation seems to take place within the spacer because its middle and right BamHI endonuclease fragments are preferentially transcribed, whereas only few RNA sequences complementary to the left spacer fragment are found. It is concluded that at least some spacer regions contain promoters for transcription and can be transcribed either into a special class of "spacer transcripts" or into molecules covalently linked to rRNA precursor.

Animals↗
PubMed1978

DNAs of simian virus 40 and polyoma direct the synthesis of viral tumor antigens and capsid proteins in Xenopus oocytes.

Purified simian virus 40 and polyoma DNAs injected into nuclei of Xenopus oocytes were transcribed and subsequently translated into virus-specific tumor antigens and capsid proteins. Simian virus 40 large and small tumor antigens synthesized in the oocytes were indistinguishable, by gel electrophoresis and [35S]methionine-labeled tryptic peptide mapping, from the corresponding polypeptides synthesized in CV-1 African green monkey cells. The synthesis of large simian virus 40 tumor antigen implies the correct splicing of its mRNA, which is complementary to nonadjacent nucleotide sequences in the early region of the viral genome. Polyoma DNA directed synthesis of two polyoma tumor antigen polypeptides, 57,000 Mr and small tumor antigen, and of the main capsid protein.

Animals↗