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T Dingermann

Publications and source records attributed to T Dingermann.

At least 55 records · Page 3Linked to original sources
PubMed1996

Developmentally regulated promoters from Dictyostelium discoideum as molecular markers for testing potential teratogens.

Already very early in the course of the development of new pharmaceutically relevant drugs toxicological tests are most important. In addition to acute and chronic toxicity the estimation of the teratogenic potential is rather crucial. We have recently shown that the eukaryotic microorganism Dictyostellium discoideum is a useful organism to test the cytotoxicity of chemical compounds. Since D. discoideum is competent of undergoing both vegetative growth and development, further investigations were aimed to establish a D. discoideum-based test system which could predict possible interference of drugs with developmental programs. We developed a method which allows to detect and to quantify effects of possible teratogens on D. discoideum development. This method is based on different transgenic D. discoideum strains, each carrying a bacterial lacZ gene under the control of a distinct developmentally regulated D. discoideum promoter. Here we describe the effects of the known teratogenic compound valproic acid (VPA) on this system.

Animals↗
PubMed1995

Analysis of gene function in Dictyostelium.

Over the past ten years, powerful molecular genetic techniques have been developed to analyze gene function in Dictyostelium. DNA-mediated transformation using a variety of selections and vectors has allowed the introduction of wild-type or modified genes that are under various forms of transcriptional control. Homologous recombination is efficient and can be used to modify the genome in precise ways. In addition, it is now possible to clone genes based on their mutant phenotype alone, either by insertional mutagenesis, or by screening antisense expression cDNA libraries. Finally, a nearly complete physical map of the genome is available and so genes are easily mapped by physical techniques. We discuss many of these advances within the context of major research problems presently under study.

Animals↗
PubMed1995

Expression of the human muscarinic receptor gene m2 in Dictyostelium discoideum.

We have expressed a functional human muscarinic M2 receptor, under the control of the homologous discoidin I gamma promoter, in the cellular slime mold Dictyostelium discoideum. The use of a contact site A leader peptide ensured insertion of the newly synthesized receptor protein into the plasma membrane. Due to the characteristics of the discoidin I gamma promoter, the M2 receptor is expressed during late growth and early development. The heterologously expressed M2 receptors show binding characteristics similar to authentic receptors. Membranes as well as whole cells can be used in ligand binding assays.

Amino Acid Sequence↗
PubMed1995

Expression of CSA-hm2 fusion in Dictyostelium discoideum under the control of the Dictyostelium ras promoter reveals functional muscarinic receptors.

We have expressed the human m2 muscarinic receptor gene in the cellular slime mold Dictyostelium discoideum. Expression under the control of the constitutive actin 6 promoter without a D. discoideum leader peptide results in cells which seem to respond to muscarinic agonists initially, but which quickly revert to non responding cells only after a few generations. However, when expressing the hm2 gene as a fusion gene together with the CSA leader peptide under the control of the regulated D. discoideum ras promoter cells are obtained which express functional muscarinic M2 receptors in a stable manner. As expected from the typical regulation of the ras promoter, M2 receptors are expressed only during development. In ligand binding assays these heterologously expressed receptors show binding characteristics similar to authentic M2 receptors.

Animals↗
PubMed1994

Isolation of transcription factor IIIC from Dictyostelium discoideum.

Transcription factor IIIC (TFIIIC) binds in a sequence-specific manner to RNA-polymerase-III-transcribed genes (e.g. tRNA genes). It sequesters other transcription factors into the preformed complex, thereby activating transcription by RNA polymerase III. The Dictyostelium discoideum homologue of TFIIIC was highly purified by affinity chromatography based on its tDNA-binding activity. This TFIIIC homologue is a multicomponent factor (molecular mass 380 kDa), which binds to the B-box element of the internal tRNA gene promoter without significant A-box interaction. Partially purified D. discoideum TFIIIC is able to functionally complement a human RNA polymerase III in vitro transcription system depleted of human TFIIIC. We provide evidence that partially purified D. discoideum TFIIIC interacts in vitro with gene-external B-box elements present down-stream of many D. discoideum tRNA genes.

Animals↗
PubMed1994

Internally located and oppositely oriented polymerase II promoters direct convergent transcription of a LINE-like retroelement, the Dictyostelium repetitive element, from Dictyostelium discoideum.

The Dictyostelium discoideum NC4 genome harbors approximately 150 individual copies of a retrotransposable element called the Dictyostelium repetitive element (DRE). This element contains nonidentical terminal repeats (TRs) consisting of conserved building blocks A and B in the left TR and B and C in the right TR. Seven different-sized classes of RNA transcripts from these elements were resolved by Northern (RNA) blot analysis, but their combined abundance was very low. When D. discoideum cells were grown in the presence of the respiratory chain blocker antimycin A, steady-state concentrations of these RNA species increased 10- to 20-fold. The D. discoideum genome contains two DRE subtypes, the full-length 5.7-kb DREa and the internally deleted 2.4-kb DREb. Both subtypes are transcribed, as confirmed by analysis of cloned cDNA. Primary transcripts from the sense strand originate at nucleotide +1 and terminate at two dominant sites, located 21 or 28 nucleotides upstream from the 3' end of the elements. The activity of a reasonably strong polymerase II promoter in the 5'-terminal A module is slightly upregulated by the tRNA gene located 50 +/- 4 nucleotides upstream and drastically reduced by the adjacent B module of the DRE. Transcripts from the opposite DNA strand (complementary-sense transcripts) were also detected, directed by an internally located polymerase II promoter residing within the C module. This latter transcription was initiated at multiple sites within the oligo(dA12) stretch which terminates DREs.

Animals↗
PubMed1994

Secretion of interleukin-6 by human meningioma cells: possible autocrine inhibitory regulation of neoplastic cell growth.

Using cell culture techniques, the authors have previously shown that human meningioma cells secrete an autocrine growth stimulator related to platelet-derived growth factor. Here, they further demonstrate potential autocrine inhibitory regulation of meningioma cell growth by interleukin (IL)-6. Constitutive IL-6 production was detected in all meningiomas studied, in the form of protein as well as IL-6-specific messenger ribonucleic acid. The IL-6 immunoreactivity in conditioned medium from three different meningioma cultures eluted from a Sephadex G-100 column was evidenced by a single peak corresponding to a molecular weight of about 32 kD. Interleukin-6 secretion was remarkably stimulated by tumor necrosis factor-alpha, IL-1 beta, and IL-4, and was also influenced by a combination of epidermal growth factor and bromocriptine. Recombinant IL-6 exhibited a significant dose-dependent inhibitory effect on meningioma cell proliferation. The maximum effect was observed at concentrations of 10 to 100 pg/ml, with the decrease in thymidine incorporation ranging from 21% to 35% versus control. Addition of an anti-IL-6 antibody enhanced the growth-stimulating effect of meningioma-derived conditioned medium. The rate of IL-6 secretion tended to show an inverse correlation with meningioma growth rate. The results presented here and the previous results suggest that the regulation of meningioma cell proliferation is defined by a complex network of autocrine stimulation, autocrine inhibition, and influences from multiple exogenous factors.

Blotting, Northern↗
PubMed1994

A simple cytotoxicity assay using the eucaryotic microorganism Dictyostelium discoideum.

First attempts to evaluate the potential of the cellular slime mold Dictyostelium discoideum as a model system for cytotoxic tests of potential drugs are described. Using the cell counter and analyser system, CASY 1, two parameters, cell count and mean cell volume, could be established as relevant parameters to estimate cytotoxicity. The effects on these two parameters of two well characterized drugs, the gentamycin analogue G418 and doxorubicin, are reported.

Animals↗
PubMed1994

Characterization of transcripts from the Dictyostelium discoideum retrotransposable genetic element DRE.

All of the approximately 150 copies of the Dictyostelium discoideum retrotransposable element DRE are integrated 50 +/- 4 nucleotides upstream from different transfer RNA genes. These genomic regions are generally devoid of informative DNA why integration of the retrotransposon at these position never causes phenotypic mutations. This property makes DRE attractive as a tool in gene therapy where vectors with predictable integration specificities are still not available. Due to the nature as retrotransposon transcription is a prerequisite for the element to integrate at a new genomic position. Here we describe some transcription properties of DRE based on Northern blot analyses and on the characterization of in vitro synthesized cDNAs.

Animals↗
PubMed1993

Different organization of the tRNA-gene-associated repetitive element, DRE, in NC4-derived strains and in other wild-type Dictyostelium discoideum strains.

The retrotransposon DRE (Dictyostelium repetitive element) was discovered in the course of an extensive study concerning the genomic organization of tRNA genes in the NC4-derived strains AX2 and AX3 of the cellular slime mold Dictyostelium discoideum. As a striking feature, DRE was found exclusively in a constant orientation and at a constant distance upstream from different tRNA genes. About 150-200 DRE with intact 5'-terminal-repeat structures are present in NC4-derived strains. These strains were termed high-copy DRE strains (HCD strains) as opposed to low-copy DRE strains (LCD strains) such as the wild-type D. discoideum isolates DD61, WS380B, OHIO and V12. LCD strains contain only 3-15 DRE with intact 5'-terminal-repeat-structures. However, in addition to these few intact elements, many 5'-truncated DRE elements are present in LCD strains. In HCD strains, most DRE show typical structural characteristics of retrotransposons containing terminal repeats at both ends, which seems to be one prerequisite for active transposition. In LCD strains, however, most DRE elements are 5'-truncated, which is a common feature of eukaryotic LINE elements. Despite their truncated 5'-ends, DRE in LCD strains retain unique integration specificities, i.e. they are always found position-specifically and orientation-specifically integrated in front of tRNA genes, flanked by a 12-16-bp target-site duplication.

Animals↗
PubMed1993

The Dictyostelium discoideum 5S rDNA is organized in the same transcriptional orientation as the other rDNAs.

In eukaryotes 5S rRNA genes are transcribed by RNA polymerase III. These genes occur in D. discoideum on the ca. 90 copies of an extrachromosomal palindrom together with the other ribosomal RNAs, which are generally transcribed by RNA polymerase I. A 5S rRNA gene has been isolated and its nucleotide sequence as well as the organization of the gene relative to the RNA polymerase I operon has been determined. The sequence of the coding region corresponds exactly to an earlier published 5S rRNA sequence. The genes are located just downstream from the 26S RNA and transcription orientations of the pol I genes and the pol III gene point into the same direction, away from the centromer of the palindrom. The isolated gene appears to be functional since it serves as a specific target for a nuclear protein, most likely TFIIIA. A genomic copy of a 5S rRNA pseudogene has been isolated from the D. discoideum strain V12. This pseudocopy contains nucleotides 52 to 82 of a bona fide 5S rRNA gene with only three mismatches. It resides 78 nucleotides downstream from the glu13(UUC) tRNA gene which in the D. discoideum strain V12 is associated with the retrotransposable element DRE.

Animals↗
PubMed1993

Structure of the promoter region of the gene encoding cytochrome c oxidase subunit V in Dictyostelium.

A cDNA for the nuclear-encoded subunit V of Dictyostelium discoideum cytochrome-c oxidase was used as a probe to screen a genomic library and isolate the complete gene. Primer-extension analysis revealed two transcription start sites located 32 and 39 nucleotides upstream of the translation initiation codon. The chloramphenicol acetyltransferase assay in transient and stable Dictyostelium transformants indicated that the 400-bp dT-rich segment 5' to the transcription start sites retained promoter activity. This region contains an octanucleotide sequence similar to the yeast HAP2/3/4 responsive element.

Animals↗
PubMed1993

Nucleotide sequence of a Dictyostelium discoideum gene encoding a protein homologous to the yeast ribosomal protein S31.

A cDNA clone has been isolated whose coding potential is significantly homologous to the yeast ribosomal protein S31. The single copy genomic gene contains a 271 bp intron immediately downstream from the ATG translation initiation codon and is flanked by cannonical exon/intron junctions. The intron carries a CAATCAAT motif which has been described as inducer element for discoidin I gamma expression and which has also been found within the intron of the rp29 gene form D. discoideum. The deduced protein contains 110 amino acids and is slightly basic.

Amino Acid Sequence↗
PubMed1992

Two distinct subforms of the retrotransposable DRE element in NC4 strains of Dictyostelium discoideum.

Approximately 2% of the Dictyostelium discoideum genome consists of multiple copies of a retrotransposable element termed DRE (Dictyostelium Repetitive Element). These elements have always been found integrated in a position and orientation-specific manner 50 +/- 4 nucleotides upstream of the coding region of tRNA genes (tDNAs). An intact DRE is 5.7 kb long. It carries an extensive coding region flanked by non-identical long terminal repeats (LTRs), composed of three distinct modules A, B and C. The left LTR proximal to the tRNA gene contains one or several A-modules followed by a single B-module (AnB). By contrast, the right LTR is composed of a B-module followed by a C-module (BC). Approximately 50% of the DRE elements in NC4 derivatives of D. discoideum are structurally different from the 5.7 kb DRE described above. They carry the following alterations: a) a 3.1 kb deletion in the coding region; b) two small deletions of 8 and 29 nucleotides in the B-module of the right LTR; c) a 72 bp deletion in the B-C junction; and d) three distinct point mutations within the A-module of the left LTR. The deletion in the open reading frame encompasses the putative coding regions for reverse transcriptase adn integrase. At least 60 copies of this smaller 2.4 kb DRE subtype are found in the genome of D. discoideum NC4 strains associated with tRNA genes. Thus, inspite of their lack in reverse transcriptase and integrase those 2.4 kb elements are presumably transposable and at least all isolated copies are found exclusively in the proximity of tRNA gene loci. The enzymes needed for their replication and transposition are likely to be provided by the intact 5.7 kb DREs.

Animals↗
PubMed1992

RNA polymerase III catalysed transcription can be regulated in Saccharomyces cerevisiae by the bacterial tetracycline repressor-operator system.

We have investigated whether the RNA polymerase III-driven transcription of eukaryotic tRNA genes can be regulated by the prokaryotic tetracycline operator-repressor system. The bacterial tet operator (tetO) was inserted at two different positions (-7 and -46) upstream of a tRNA(Glu) (amber) suppressor gene. Both constructs are transcribed in Saccharomyces cerevisiae and yield functional tRNAs as scored by suppression of an amber nonsense mutation in the met8-1 allele. Controlled expression of Tet repressor was achieved by fusing the bacterial tetR gene to the yeast gal1 promoter. This leads to expression of Tet repressor in yeast on galactose--but not on glucose--containing media. Regulation of the su-tRNA gene with the tetO fragment inserted at position -7 has been demonstrated. Under conditions which allow tetR expression, cells exhibit a met- phenotype. This methionine auxotrophy can be conditionally reverted to prototrophy by adding tetracycline. However, a su-tRNA gene with the tetO fragment inserted at position -46 cannot be repressed. Our results demonstrate clearly that the bacterial repressor protein binds to its operator in the yeast genome. Formation of this complex in the vicinity of the pol III transcription initiation site reduces the level of su-tRNA at least 50-fold as concluded from quantitative primer extension analyses. This indicates for the first time that class III gene expression can be regulated by a DNA binding protein with its target site in the 5'-flanking region and that a prokaryotic repressor can confer regulation of a suitably engineered tRNA gene.

Alleles↗