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R Stalder

Publications and source records attributed to R Stalder.

At least 19 recordsLinked to original sources
PubMed1999

CLIP-170 highlights growing microtubule ends in vivo.

A chimera with the green fluorescent protein (GFP) has been constructed to visualize the dynamic properties of the endosome-microtubule linker protein CLIP170 (GFP-CLIP170). GFP-CLIP170 binds in stretches along a subset of microtubule ends. These fluorescent stretches appear to move with the growing tips of microtubules at 0.15-0.4 microm/s, comparable to microtubule elongation in vivo. Analysis of speckles along dynamic GFP-CLIP170 stretches suggests that CLIP170 treadmills on growing microtubule ends, rather than being continuously transported toward these ends. Drugs affecting microtubule dynamics rapidly inhibit movement of GFP-CLIP170 dashes. We propose that GFP-CLIP170 highlights growing microtubule ends by specifically recognizing the structure of a segment of newly polymerized tubulin.

Animals↗
PubMed1998

Target specificity of insertion element IS30.

The Escherichia coli resident mobile element IS30 has pronounced target specificity. Upon transposition, the element frequently inserts exactly into the same position of a preferred target sequence. Insertion sites in phages, plasmids and in the genome of E. coli are characterized by an exceptionally long palindromic consensus sequence that provides strong specificity for IS30 insertions, despite a relatively high level of degeneracy. This 24-bp-long region alone determines the attractiveness of the target DNA and the exact position of IS30 insertion. The divergence of a target site from the consensus and the occurrence of 'non-permitted' bases in certain positions influence the target activity. Differences in attractiveness are emphasized if two targets are present in the same replicon, as was demonstrated by quantitative analysis. In a system of competitive targets, the oligonucleotide sequence representing the consensus of genomic IS30 insertion sites proved to be the most efficient target. Having compared the known insertion sites, we suppose that IS30-like target specificity, which may represent an alternative strategy in target selection among mobile elements, is characteristic of the insertion sequences IS3, IS6 and IS21, too.

Binding Sites↗
PubMed1997

Mutations in the carboxy-terminal part of IS30 transposase affect the formation and dissolution of (IS30)2 dimer.

The transposase of IS30 catalyses different transpositional rearrangements via the dimer (IS30)2 intermediate structure. Mutation analysis provides evidence that the C-terminal part of IS30 transposase is required for the formation and dissolution of (IS30)2 dimer. C-terminal mutants are also defective in transpositional fusion; however, this deficiency can be 'suppressed' by addition of the final product of site-specific dimerisation, the core (IS30)2 intermediate structure. The transposase part studied shows significant homologies in three highly conserved regions to proteins of IS30-related mobile elements.

Amino Acid Sequence↗
PubMed1993

Formation of the tandem repeat (IS30)2 and its role in IS30-mediated transpositional DNA rearrangements.

Plasmids carrying two IS30 elements in the same orientation, as in the composite transposon Tn2706, are structurally unstable in Escherichia coli. A primary segregation product is formed by site-specific deletion of the sequences carried between the two IS30 elements. The resulting covalently closed replicon carries the two IS30 elements as tandem repeats separated by only 2 bp. This (IS30)2 structure is extremely unstable, but it can nevertheless be isolated on its vector plasmid and, after purification, can be reintroduced into host cells by transformation. Among the descendants of transformants of recA- bacteria, replicated copies of the introduced (IS30)2 structure are still present, together with various kinds of segregation products which provide evidence for the efficient generation of DNA rearrangements. Most abundant is the product of another site-specific recombination between two identical ends of the IS30 elements involved, which results in the presence of just one intact IS30 on the plasmid. Apart from this, and depending on the presence of appropriate targets for IS30 transposition, various transposition products of (IS30)2 are also seen. Intramolecular reactions lead to DNA inversions and deletions with breakpoints other than IS30 ends. In intermolecular reactions inverse transposition occurs at high frequency and one also obtains simple transposition and cointegration. A mutational study revealed the requirement in cis of one intact IS30 transposase gene and of both proximal ends of the two IS30 elements concerned not only for the formation of (IS30)2, but also for its further rearrangement reactions, including the efficient formation of site-specific deletions. A model is proposed, which postulates that (IS30)2 intermediates play a key role in IS30 transposition pathways in which the formation of (IS30)2 may be rate-limiting. Once this structure is formed, it gives rise to a burst of transpositional rearrangements in the subclone carrying (IS30)2. Evolutionary implications of these findings are discussed.

Base Sequence↗
PubMed1992

RPC82 encodes the highly conserved, third-largest subunit of RNA polymerase C (III) from Saccharomyces cerevisiae.

RNA polymerase C (III) promotes the transcription of tRNA and 5S RNA genes. In Saccharomyces cerevisiae, the enzyme is composed of 15 subunits, ranging from 160 to about 10 kDa. Here we report the cloning of the gene encoding the 82-kDa subunit, RPC82. It maps as a single-copy gene on chromosome XVI. The UCR2 gene was found in the opposite orientation only 340 bp upstream of the RPC82 start codon, and the end of the SKI3 coding sequence was found only 117 bp downstream of the RPC82 stop codon. The RPC82 gene encodes a protein with a predicted M(r) of 73,984, having no strong sequence similarity to other known proteins. Disruption of the RPC82 gene was lethal. An rpc82 temperature-sensitive mutant, constructed by in vitro mutagenesis of the gene, showed a deficient rate of tRNA relative to rRNA synthesis. Of eight RNA polymerase C genes tested, only the RPC31 gene on a multicopy plasmid was capable of suppressing the rpc82(Ts) defect, suggesting an interaction between the polymerase C 82-kDa and 31-kDa subunits. A group of RNA polymerase C-specific subunits are proposed to form a substructure of the enzyme.

Amino Acid Sequence↗
PubMed1991

The correlation of body growth with diethylnitrosamine-induced hepatocarcinogenesis in relation to serum insulin and somatomedin-C.

Caloric restriction depresses the development of several types of tumours, yet the mechanisms involved are poorly understood. In the present experiment we investigated the development of diethylnitrosamine (DEN)-induced liver tumours in mice treated with caffeine. The latter was found to reduce body growth, possibly due to increased energy expenditure, without reducing food consumption. Newborn mice received an i.p. injection of DEN. At weaning they were either fed lab chow ad libitum, with the same diet containing 0.2% (w/w) of caffeine, or their access to food was restricted to 70% of that consumed by the ad libitum group. Diet caloric restriction starting at weaning in male Swiss mice decreased the rate of development of glucose-6-phosphatase-deficient (G6Pd) preneoplastic foci. At the age of 24 weeks, 10% of the surface of a standardized liver section of ad libitum fed mice was G6Pase negative, compared to only 1% in the restricted mice due to a reduction of the number and size of these preneoplastic foci. The number and size of G6Pd foci decreased to the same extent with the ingestion of a lab chow supplemented with 0.2% of caffeine as with the diet restriction. This finding suggests that restriction slows down hepatic tumour growth by modifying body growth rather than by limited nutrient supply. In parallel, somatomedin-C (Sm-C) and insulin secretion following glucose challenge were decreased in diet restricted mice and those treated with 0.2% caffeine. The serum Sm-C and insulin levels were respectively 480 and 4.6 ng/ml in the restricted mice, 519 and 16.6 ng/ml in the caffeine-fed mice and 664 and 25.7 ng/ml in the ad libitum fed mice. Our results suggest that the decrease of secretion of these two hormones that are known mitogens for hepatocytes in vitro may be responsible at least in part for the reduction in the growth of liver tumours.

Animals↗
PubMed1991

The effects of alternating dietary restriction and ad libitum feeding of mice on the development of diethylnitrosamine-induced liver tumours and its correlation to insulinaemia.

The effects of alternating ad libitum feeding and 30% restriction of the dietary intake on the development of diethylnitrosamine (DEN)-induced hepatic neoplasia were investigated. Dietary restriction retarded the growth of glucose-6-phosphatase-deficient (G6Pd) preneoplastic foci and subsequently that of hepatocellular adenomas and adenocarcinomas. The number of foci in standardized liver sections increased from 4.44 foci/cm2 at 12 weeks to 9.65 foci/cm2 at 24 weeks in ad libitum fed animals but only from 2.35 foci/cm2 to 3.29 foci/cm2 in restricted animals. In animals fed first ad libitum for 12 weeks and then for 12 weeks on a restricted diet, the number of G6Pd foci dropped from 4.44 at 12 weeks to 3.54 at 24 weeks. This reduction appeared to be the result of a regression of the small sized G6Pd foci. Dietary restriction was most efficient in inhibiting the development of G6Pd foci when started early in life. Conversely, the growth of foci was stimulated when the mice first had restricted access to food and thereafter were fed ad libitum. The plasma insulin concentrations after a glucose challenge increased with age. Insulinaemia was much higher in ad libitum fed compared to the restricted mice. It was correlated to the number of G6Pd foci in the liver. This study suggests that insulin, which is a known mitogen for hepatocytes in vitro, may contribute to the promotion of DEN-induced liver tumours in mice.

Adenoma↗
PubMed1990

Preparation and characterization of yeast nuclear extracts for efficient RNA polymerase B (II)-dependent transcription in vitro.

We present a reproducible method for the preparation of nuclear extracts from the yeast Saccharomyces cerevisiae that support efficient RNA polymerase B (II)-dependent transcription. Extracts from both a crude nuclear fraction and Percoll-purified nuclei are highly active for site-specific initiation and transcription of a G-free cassette under the Adenovirus major late promoter. At optimal extract concentrations transcription is at least 5 times more efficient with the yeast extracts than with HeLa whole cell extracts. We show that the transcriptional activity is sensitive to alpha-amanitin and to depletion of factor(s) recognizing the TATA-box of the promoter. The in vitro reaction showed maximal activity after 45 min, was very sensitive to Cl-, but was not affected by high concentrations of potassium. We find that the efficiency of in vitro transcription in nuclear extracts is reproducibly high when spheroplasting is performed with a partially purified beta 1,3-glucanase (lyticase). Therefore a simplified method to isolate the lyticase from the supernatant of Oerskovia xanthineolytica is also presented.

Amanitins↗
PubMed1990

A yeast homolog of the human UEF stimulates transcription from the adenovirus 2 major late promoter in yeast and in mammalian cell-free systems.

We report the identification and purification of a yeast factor functionally homologous to the human upstream element factor (UEFh). Although the yeast protein (UEFy) has a higher molecular weight than the HeLa UEF (60 kD versus 45 kD) both have identical DNA-binding properties: the purified UEFy recognizes the Adenovirus 2 (Ad2) major late promoter upstream element (MLP-UE; from nucleotide -49 to -67) as well as the IVa2 upstream element (IVa2-UE; from nucleotide -98 to -122) with a higher affinity for the MLP-UE than for the IVa2-UE. Based on its DNA binding specificity, size and thermostability, the UEFy protein appears also similar or equivalent to the centromere binding protein CP1. In a competition assay with oligonucleotides containing the MLP-UE binding site, a drastic reduction of Ad2 MLP transcription was observed both in a HeLa and in a yeast cell free system, which was restored by addition of either the purified UEFh or UEFy proteins. We conclude that both UEFh and UEFy activate transcription from the Ad2 MLP upon binding to the upstream element, whatever is the in vitro cell-free system (yeast or HeLa). This indicate that some regulatory function represented by the upstream element and its cognate factor, is well conserved between human and yeast.

Adenoviruses, Human↗
PubMed1990

The N-terminal domain of the insertion sequence 30 transposase interacts specifically with the terminal inverted repeats of the element.

The gene for the insertion sequence (IS) 30 transposase is placed under the control of the tac promoter, and large quantities of transposase are expressed upon induction. The resulting protein precipitates inside the Escherichia coli cells in the form of inclusion bodies which, upon cell lysis, cannot be dissolved under nondenaturing conditions. In contrast, the N-terminal third of the transposase, a 17-kDa protein produced by a truncated gene, can be purified and is able to interact site specifically with the ends of the IS30 element. In DNase I footprint experiments, regions of 26 nucleotides on one DNA strand and 19 nucleotides on the other strand at either end of the element are protected from nuclease digestion. It is concluded that a functional DNA-binding domain can be formed by expression of only one-third of the complete IS30 transposase. Sequence comparison shows a homology of the IS30 ends to the ends of IS4351 and to the L1 end of bacteriophage Mu.

Amino Acid Sequence↗
PubMed1990

A carcinogenicity study of instant coffee in Swiss mice.

Commercially available regular instant coffee was given in the diet to barrier-maintained, specified pathogen-free Swiss mice for 2 yr. Groups of 150 males and 150 females were fed diets containing 10, 25 or 50 g instant coffee powder/kg. The animals had already been exposed to coffee in utero. Coffee increased the energy expenditure of the animals as shown by increased daily calorific intake and depressed growth. The overall tumour incidence was inversely correlated to the coffee intake, and no unusual tumour or site of origin was found. The most frequent neoplasms were lymphosarcomas, bronchiolo-alveolar adenomas and adenocarcinomas, as well as hepatocellular adenomas. The incidence of total neoplasms (benign and malignant) decreased from 70.6 and 56.8% in control males and females, respectively, to 34.8 and 36.2%, respectively, in the high-dose group. This decrease, which was essentially due to a reduction in the number of lymphosarcomas and hepatocellular adenomas, was associated with a slower growth rate. The number of leiomyomas in the uterus was slightly increased due to coffee intake as shown by the analysis of positive trend (P less than or equal to 0.05). However, the incidence of this benign tumour was very low; 2.72% of mice affected in the high-dose group, 1.37% in the low-dose group and 0% in the control and medium-dose groups. From this study it is concluded that instant coffee did not increase the incidence of malignant neoplasms in mice when fed at dietary levels of up to 5% for 2 yr.

Animals↗
PubMed1990

Overexpression of tubulin in yeast: differences in subunit association.

Microtubules are cytoskeletal organelles composed principally of polymerized alpha beta-tubulin heterodimers. The specific roles and the detailed structures of the individual alpha- and beta-tubulin subunits have not been established, since the conditions necessary for separating the heterodimer result in loss of the subunits' ability to repolymerize. We have overcome this obstacle by constructing plasmids which allow regulated overexpression of individual tubulin subunits in the yeast Saccharomyces cerevisiae under control of the galactose promoter. Overproduction was monitored with alpha- and beta-tubulin-specific antibodies using immunoblotting of cell extracts, and the state of association of the individual subunits in vivo was determined by immunofluorescence microscopy. Cells overproducing only beta-tubulin accumulated fibrous structures associated with the cell membrane, whereas cells overproducing only alpha-tubulin displayed a diffuse signal throughout the cytoplasm. Cells simultaneously overexpressing alpha- and beta-tubulin subunits accumulated membrane-associated, filamentous arrays in which both subunits were incorporated. When cells with the fibrous tubulin-containing structures are treated with zymolyase, a yeast cell wall disrupting enzyme, the fibers appear to splay apart, suggesting that the immunofluorescent rings represent bundles of fibers. Cells overproducing beta-tubulin alone or both alpha- and beta-tubulin were examined at various times after galactose induction, and significant differences were found in the tubulin association state prior to the formation of fibers. For alpha beta-tubulin, fibers form directly from a nuclear structure, whereas beta-tubulin alone first accumulates in the cytoplasm. The differences in patterns of tubulin accumulation and assembly presumably reflect a difference in the intrinsic association properties of the alpha- and beta-subunits.

Antibodies↗
PubMed1989

Characterization of in vitro constructed IS30-flanked transposons.

In order to facilitate functional studies on the mobile genetic element IS30, a resident of the Escherichia coli chromosome, transposon structures with two copies of IS30 flanking the chloramphenicol-resistance gene cat were constructed in vitro. Transposons containing IS30 as direct repeats (Tn2700 and Tn2702) transpose from multicopy plasmids into the genome of phage P1-15, thus giving rise to special transduction for cat with frequencies between 10(-5) and 10(-8)/plaque-forming unit. In contrast, transposon structures with IS30 in inverted repeat (Tn2701 and Tn2703) showed no detectable (less than 10(-9] transposition activity in vivo. By restriction analysis, two insertion sites of Tn2700 and Tn2702 on the phage P1-15 genome were indistinguishable from those observed earlier with a single copy of the IS30 element. These two insertion sites were used several times independently by Tn2700 and Tn2702. This confirms the non-random target selection by the element and it indicates that transposition of Tn2700 and Tn2702 follows the same rules as that of IS30.

Bacteriophages↗
PubMed1987

The influence of food intake on the development of diethylnitrosamine-induced liver tumours in mice.

The aim of this study was to determine whether a dietary restriction, which significantly decreases body weight gain, also influences the development of diethylnitrosamine (DEN)-induced liver tumours in Swiss mice. At birth, mice were injected i.p. with a single dose of DEN (0.4 mumol/g body wt); negative control mice were sham injected. After weaning the animals received a stock diet either ad libitum (control group) or at 30% restriction (restricted group). A positive control group was fed ad libitum and received phenobarbital (500 p.p.m.) in their drinking water. At 12 weeks of age, glucose-6-phosphatase (G6Pase)-deficient foci were present in the liver of 80% of the control animals but only in 32% of the restricted group. Quantitatively, restricted animals had fewer foci per unit volume liver and these were smaller than in the control animals. By 36 weeks of age, hepatocarcinomas were seen in 100% of the control mice while in the restricted group there were no such malignant lesions and only 32% showed adenomas. The results clearly show that restriction of food intake inhibits promotion and progression of induced liver tumours. Amongst other uses, this model permits the study of the effect of dietary restriction on liver tumours, at an early stage.

Animals↗
PubMed1985

CGP 22979A, a renal vasodilator with natriuretic properties.

Renal vasodilation might be an interesting new antihypertensive principle. In the present study, the prodrug approach was adopted to synthesize a preferential renal vasodilator. A hydralazine-like compound, CGP 18137A (2-hydrazino-5-n-butyl-pyridine), was substituted with an N-acetyl-gamma-glutamyl residue. The resulting derivative, CGP 22979A [N-acetyl-L-glutamic acid-N[N2-(5-n-butyl-2-pyridyl)hydrazide]], was inactive in the isolated perfused mesenteric artery, whereas the parent compound induced a dose-dependent inhibition of vasoconstrictor stimuli. When administered i.v. to anesthetized rats in doses between 1.0 and 10.0 mg/kg, CGP 22979A increased renal blood flow significantly, by up to 31% without affecting blood pressure. A dose of 4.0 mg/kg lowered renal vascular resistance by 25% but did not alter total systemic, mesenteric and iliac vascular resistance. A dose of 10.0 mg/kg increased glomerular filtration rate by up to 42%. In conscious rats, the same dose increased sodium excretion by 200%. Because CGP 22979A induces renal vasodilation in rats and has a preferential action on the afferent arterioles, it might be a useful tool for studying the antihypertensive potential of renal vasodilation.

Animals↗
PubMed1984

Structure linkage, and sequence of the two genes encoding the delta and gamma subunits of the nicotinic acetylcholine receptor.

We have cloned and sequenced a fragment of the chicken genome approximately 9 kilobases in length that comprises the genes encoding the delta and gamma subunits of the nicotinic acetylcholine receptor. The two genes are homologous and have identical structures: both consist of 12 exons, some of which precisely correspond to predicted structural domains of the receptor subunits. The delta and gamma subunit genes are encoded by the same DNA strand and are very closely linked, there being only 740 base pairs between the last codon of delta and the initiator codon of gamma. Blot analysis demonstrates that the genes we describe are unique in the genome. Comparison of the predicted protein sequence for the corresponding subunits of chicken and of the elasmobranch Torpedo reveals a high degree of conservation in some but not all of the protein domains.

Amino Acid Sequence↗
PubMed1984

The significance of vasopressin as a pressor agent.

The antidiuretic hormone, arginine-vasopressin (AVP), may participate in the regulation of blood pressure (BP) through its vasoconstrictor effects. In anesthetized rats, exogenous AVP induced stronger vasoconstriction in the mesenteric than in the renal vascular bed. Conversely, mesenteric but not renal vascular resistance was reduced by a vascular antagonist of AVP, d(CH2)5 VDAVP, in rats with increased endogenous AVP after anesthesia, dehydration, or injection of glycerol. Another vascular AVP-antagonist, d(CH2)5 Tyr (Me) AVP, induced a transient fall in BP in conscious primates (marmosets) after diuretic-induced volume depletion. In conscious rats with established deoxycorticosterone acetate (DOCA)/salt hypertension, d(CH2)5 Tyr (Me) AVP decreased systolic BP after acute administration. After chronic administration of this antagonist during 6 weeks after the beginning of DOCA/salt treatment, the severity of hypertension was reduced. When another, AVP-antagonist, d(CH2)5-D-Tyr (Et) VAVP, which blocks vascular and renal tubular AVP-receptors, was administered chronically, the development of DOCA/salt hypertension was prevented at the expense of severe and persistent hypernatremia. These results demonstrate that under certain conditions the vascular effects of AVP may contribute to the maintenance of BP, AVP appears to participate in the pathogenesis of DOCA/salt hypertension through both its vasoconstrictor and its antidiuretic effects.

Acute Kidney Injury↗