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M F Trendelenburg

Publications and source records attributed to M F Trendelenburg.

At least 19 recordsLinked to original sources
PubMed2000

Stability of DNA repeats in Escherichia coli dam mutant strains indicates a Dam methylation-dependent DNA deletion process.

In this study we report on the stabilization of short direct repetitive DNA elements. We arranged a 20 bp SK-primer element in a direct repeat manner within the cloning vector pBluescript KS (+). This resulted in an array of 27 direct repeats consisting of 24 bp units. We show that this plasmid could only be propagated without deletion of repeats in dam mutant Escherichia coli hosts, whereas all efforts to use strains that were defective in the methylation-dependent restriction system and the recA- or the mismatch repair-dependent deletion system failed. The deletions always affected whole repeat units and not parts of them, leading to an unpredictable reduction of the unit number down to a range of between 12 and two during propagation. We conclude that a Dam methylation-dependent, but recA- and mismatch repair-independent, deletion mechanism caused the DNA rearrangements without an obvious involvement of the known methylated-DNA restriction systems.

Base Sequence↗
PubMed1999

An approach to an objective background subtraction for elemental mapping with core-edges down to 50 eV: description, evaluation and application.

To image the distribution of a specific element in a specimen with an energy filtering TEM, the element-unspecific background under the core-edge has to be subtracted. The most commonly used procedure is the three-window power-law method leading to considerable systematic errors for low-energy core-edges. Here a new method is described which can be considered as a generalized difference method. Characteristic examples for element detection in biological specimens using this method are shown. The background under the core-edge can be described by one or two pre-edge windows as a polynome of third order. This function can be deduced from specimen areas that are not known to contain the element or from a second specimen used as a standard. Control experiments showed that background subtraction for on-overlapping core-edges in the low-loss region (50-200 eV) needs two pre-edge images, whereas at higher-energy losses (> 300 eV) only one pre-edge image is necessary. With the method described, objective elemental mapping becomes possible even for edges at 50-100 eV. This was proven for the M2,3-edge of iron at 60 eV. The detection of phosphorous was possible with a signal-to-noise ratio five times higher than when using the three-window method. Preliminary data showed that it should be possible to detect calcium with only one image before the edge.

Animals↗
PubMed1998

Structure and dynamics of human interphase chromosome territories in vivo.

A new approach is presented which allows the in vivo visualization of individual chromosome territories in the nuclei of living human cells. The fluorescent thymidine analog Cy3-AP3-dUTP was microinjected into the nuclei of cultured human cells, such as human diploid fibroblasts, HeLa cells and neuroblastoma cells. The fluorescent analog was incorporated during S-phase into the replicating genomic DNA. Labelled cells were further cultivated for several cell cycles in normal medium. This well-known scheme yielded sister chromatid labelling. Random segregation of labelled and unlabelled chromatids into daughter nuclei resulted in nuclei exhibiting individual in vivo detectable chromatid territories. The territories were composed of subcompartments with diameters ranging between approximately 400 and 800 nm which we refer to as subchromosomal foci. Time-resolved in vivo studies demonstrated changes of positioning and shape of territories and subchromosomal foci. The hypothesis that subchromosomal foci persist as functionally distinct entities was supported by double labelling of chromatin with CldU and IdU, respectively, at early and late S-phase and subsequent cultivation of corresponding cells for 5-10 cell cycles before fixation and immunocytochemical detection. This scheme yielded segregated chromatid territories with distinctly separated subchromosomal foci composed of either early- or late-replicating chromatin. The size range of subchromosomal foci was similar after shorter (2 h) and longer (16 h) labelling periods and was observed in nuclei of both living and fixed cells, suggesting their structural identity. A possible functional relevance of chromosome territory compartmentalization into subchromosomal foci is discussed in the context of present models of interphase chromosome and nuclear architecture.

Bromodeoxyuridine↗
PubMed1997

Reassembly of functional nucleoli following in situ unraveling by low-ionic-strength treatment of cultured mammalian cells.

In order to determine the most persistent components of the nucleolus that might serve as "core" nucleolar elements, we studied the reactivity of nucleoli in living mammalian cells subjected to hypotonic buffer saline followed by the incubation of the cells in an isotonic medium. To document as precisely as possible the fine structural changes which occurred, the cells were examined by video-enhanced optical microscopy, fluorescence confocal laser scanning microscopy, and electron microscopy combined with cytochemistry. Light microscopic autoradiography was used to demonstrate the transcriptional characteristics of the reassembled nucleoli. It was shown that all the major compartments of the intact nucleolus could be substantially affected by reduction of the osmolarity of the environmental media. The dynamic events of the nucleolar unraveling in low-salt buffers occurred in the following order: dispersion of the nucleolar pars granulosa, disassociation of the fibrillar complexes into discrete fibrillar centers (FCs) and the dense fibrillar component (DFC), and the almost complete unraveling of the DFC and FCs. At the terminal stages of nucleolar dispersion, the nuclear interior was mainly composed of a loose filamentous meshwork, and none of the typically discerned nucleolar constituents was recognized. Nevertheless, when hypotonically treated cells were returned to isotonic conditions, the nucleolar bodies rapidly began to reassemble. Within 1-2 h of cell incubation under isotonicity, the nucleoli not only became clearly visible, but also reconstituted to their initial size, shape, and position within the nucleus. The ultrastructure and functional activity of the reassembled nucleoli were also found to be fully comparable to those of the untreated controls. These data indicate that the architectural composition of the interphase nucleolus is strictly controlled by the cell. As far as could be determined, none of the usual substructures of the intact nucleolus that could be substituted by complete reassembly of the nucleolar bodies in normotonic conditions, including FCs and the DFC, remained clearly preserved in the terminal stage of nucleolar unraveling. We concluded that the integrity of the nucleolus was mainly preserved by the nuclear or nucleolar matrix system rather than by any other nucleolar structural domains.

Animals↗
PubMed1997

Experimental induction of prenucleolar bodies (PNBs) in interphase cells: interphase PNBs show similar characteristics as those typically observed at telophase of mitosis in untreated cells.

Recently, it was shown that a short exposure of living mammalian cells to low ionic strength buffers (hypotonic shock) caused partial or almost complete unraveling of interphase nucleoli. However, when the cells were released from the hypotonic shock and transferred to normal isotonic medium, functionally active and structurally integral nucleoli were reassembled at their initial positions within interphase nuclei. Here, we show further that this process is accompanied by the appearance of numerous discrete extranucleolar bodies, which have striking similarities to the prenucleolar bodies (PNBs) observed in untreated cells at telophase of mitosis. (1) Like PNBs at mitosis, hypotonically induced interphase PNBs are composed of RNA-positive granules and fibrils, contain the major nucleolar protein B23 and silver-binding proteins, but lack DNA and RNA polymerase I transcription factor UBF. (2) As for mitotic PNBs, disappearance of the interphase PNB counterparts coincides with the increase in size of reconstructed nucleoli. (3) Addition of actinomycin D does not prevent assembly of interphase PNBs, but does arrest their coalescence with the chromosomal nucleolus-organizing regions and blocks the complete reformation of nucleoli. It is concluded that the assembly of PNBs generally observed at telophase of mitosis can be induced experimentally in nuclei of interphase mammalian cells in vivo. At interphase, this process is probably initiated by changes in the intracellular ionic environment.

Animals↗
PubMed1997

Transmission X-ray microscopy of intact hydrated PtK2 cells during the cell cycle.

Transmission X-ray microscopy makes it possible to investigate biological specimens, i.e. cells and organelles, in their natural wet environment. The main processes determining the contrast in X-ray microscopy are photoelectric absorption and phase shift. X-ray microscopic experiments can therefore be carried out in both amplitude and phase contrast. The Göttingen X-ray microscope at the BESSY storage ring in Berlin is described. PtK2 cells were examined during different stages of the cell cycle. All major constituents of the mitotic apparatus, e.g. chromosomes, centromeres, microtubules and centrosomes, could be visualized, as well as the main structural compartments and organelles of the interphase cell, e.g. nuclear membrane, interphase chromatin, nucleolus and cytoplasmic mitochondria, as well as parts of the cytoskeletal apparatus. In this way new information can be obtained with regard to the ultrastructure of the constituents of intact and unstained cells at a resolution which bridges the gap between light microscopy and electron microscopy. The prospects for the future application of transmission X-ray microscopy in biomedical research are discussed.

Animals↗
PubMed1997

Cell cycle-dependent translocations of a major nucleolar phosphoprotein, B23, and some characteristics of its variants.

A major nucleolar phosphoprotein, B23, is thought to play several apparently unrelated roles and appears to be associated with other cell compartments besides the nucleolus. However, characteristic properties of B23 variants still remain to be established. Here, we raised a new monoclonal antibody against B23 (20B2) and used it to address the issue particularly focusing on the events during mitosis. Also, we made an attempt to generalize the data on the cell cycle-dependent translocations of B23 by the use of three mammalian cell lines (HeLa, PK, RAMT) which were found to be immunoreactive for 20B2. In all the cell strains studied, B23 was chiefly located within the nucleolus at interphase, and was associated with a few cellular domains during mitosis. They were: the nucleoplasm (at prophase before the nuclear envelope breakdown), the cytoplasm (from prometaphase until mid telophase), the perichromosomal layer (from prometaphase till early telophase), cytoplasmic B23-containing bodies (at anaphase and telophase) and prenucleolar bodies, PNBs (at telophase). On Western blots, electrophoretic mobility of B23 was found to be the same at G1, S and G2 periods of interphase, but became slower at mitosis. In situ and cell extraction experiments showed that like the nucleolar B23, B23 of the perichromosomal layer and that of PNBs was highly resistant to extraction with Triton X-100, but could be released with Triton X-100/RNase A. These findings indicated that these portions of B23 were most likely to be associated with RNA. The cytoplasmic B23 was the major intracellular variant of B23 during mitosis. It had a slightly lower electrophoretic mobility than the perichromosomal B23 and could readily be extracted with Triton X-100 without addition of RNase A, a fact indicating that the cytoplasmic B23 was mainly in an RNA-free state. Mitosis-like translocations of B23 from the nucleolus to the nucleoplasm induced by actinomycin D increased its extractability but did not affect the electrophoretic mobility. The phosphorylation status of different B23 variants at interphase and mitosis both in controls and following the drug, is discussed.

3T3 Cells↗
PubMed1996

Multiparameter microscopic analysis of nucleolar structure and ribosomal gene transcription.

A survey of novel microscopic approaches for structural and functional analysis of subnucleolar compartments will be presented. Research on nucleolar structure and function concentrates predominantly on two distinct types of nucleoli: (1) nucleoli present during the interphase of the cell cycle in somatic tissue culture cells and (2) nucleoli present in meiotic cells, e.g. oocytes of amphibians. These nucleoli are found during meiotic prophase of oogenesis and are functional during several months of the diplotene stage of oogenesis. A further characteristic is the fact that these nucleoli are extrachromosomal, since they originate by selective ribosomal DNA (rDNA) amplification during the early pachytene stage of oogenesis. Miller-type chromatin spread preparations using transcriptionally active nucleoli, to a major part, contributed to our understanding of the structural organization of polymerase I directed pre-rRNA transcription. Although the structural organization of the template-associated pre-rRNA transcript is known in some detail from chromatin spreads, relatively little is known about structural aspects of pre-rRNA processing. In order to investigate this intriguing question in more detail, we have developed a computer-based densitometry analysis of both template-associated and template-dissociated pre-rRNA transcripts in order to follow the structural modification of pre-rRNA transcripts during processing. Another line of experiments is devoted to the in situ structure of actively transcribing genes in the nucleolus. In order to bridge the gap between light microscopy and electron microscopy we started video-enhanced light microscopical analysis of actively transcribing genes. Although the dimensions of individual spread genes are critical for detection by optical microscopy, we succeeded in obtaining the first series of images of transcribing genes in their "native' hydrated state. An additional promising type of microscopy is transmission X-ray microscopy. Recent progress in instrumentation as well as in sample preparation has allowed us to obtain the first images of density distribution within intact, fully hydrated nucleoli using amplitude-contrast and/or phase-contrast X-ray microscopy of non-contrasted, fully hydrated nucleoli at different states of transcriptional activity. Whereas the above mentioned investigations using video microscopy and X-ray microscopy are predominantly applicable to the analysis of amplified nucleoli in amphibian oocytes, which are characterized by an extremely high transcription rate of 80-90% of rDNA genes per individual nucleolus, structural analysis of the in situ arrangement of actively transcribing genes in somatic nucleoli as present in the interphase nucleus is far more difficult to perform, mainly due to the much lower number of simultaneously transcribed active genes per individual nucleolus. Visualization of actively transcribed gene clusters is approached by an integrated experimental assay using video microscopy, confocal laser scan microscopy, and antibodies against specific nucleolar proteins.

Animals↗
PubMed1996

Spatial arrangement of intra-nucleolar rDNA chromatin in amplified Xenopus oocyte nucleoli: structural changes precede the onset of rDNA transcription.

Amphibian oocyte nucleoli are a particular suited object for research on nucleolar chromatin organization. By selective rDNA amplification each pachytene oocyte nucleus accumulates 30 pg of extrachromosomal rDNA, this amount corresponds to 2 million rDNA copies. Following the selective amplification stage, the amplified gene copies are finally distributed within more than thousand extrachromosomal nucleoli per individual oocyte nucleus. The aim of the present study was first to obtain a precise documentation of the fate of amplified rDNA during early Xenopus oogenesis until the final functional integration of these copies into individual oocyte nucleoli, and, second, a close correlation of the structural data with determination of rDNA transcription rates by S1 transcript analysis for the subsequent stages of oocyte differentiation. In order to investigate the structural complexity of the intranuclear rDNA translocation process in detail, a confocal laser scan microscope (CLSM) was used, equipped with an external UV-laser. This instrumentation unambiguously allowed (i) the detection of small clusters of rDNA copies and (ii) the precise spatial documentation of the intranuclear position of rDNA clusters in relation to the protein-free pre-nucleolar protein bodies, a specific characteristic of late pachytene/early diplotene amphibian oocyte nuclei. Our results indicate that the major rDNA translocation processes, e.g. the association of rDNA clusters with pre-nucleolar protein bodies, the formation of ribbon-like pre-nucleolar units sensu Van Gansen and Schramm (J. Cell Sci. 10: 339-367, 1972), and, finally, the translocation of fused rDNA units into the interior of pre-nucleolar protein bodies, occur--for the most part--in absence of massive rDNA transcription. As shown by the S1 transcript analysis, the onset of massive rDNA transcription starts concomitantly with an unraveling of the densely packed rDNA clusters into finely dispersed rDNA units, which were shown by CLSM analysis to be distributed throughout the entire nucleolar volume.

Animals↗
PubMed1993

The RNA polymerase I-specific transcription initiation factor UBF is associated with transcriptionally active and inactive ribosomal genes.

We have characterized an anti-NOR (nucleolar organizer region) serum (P419) from a patient with rheumatoid arthritis and show that it contains antibodies directed against the RNA polymerase I-specific transcription initiation factor UBF. This serum reacts with UBF from a variety of vertebrate cells as revealed both by immunoblotting and by indirect immunofluorescence. We have used the P419 serum to study the intracellular localization of this transcription factor at the light and electron microscopic level. In interphase cells, UBF exhibits a pronounced punctate pattern and is found to be associated with necklace-like structures, which appear to reflect the transcriptionally active state of the nucleolus. Inhibition of rRNA synthetic activity caused either by nutritional starvation or by actinomycin D treatment resulted in a marked decrease in the number and in a significant increase in the size of UBF-positive granules. Under all experimental conditions applied, UBF was exclusively found within the nucleolus and was not released into the nucleoplasm or cytoplasm. During mitosis, UBF was found to be concentrated at the chromosomal NOR indicating that a significant quantity, if not all, of this factor remains bound to the ribosomal transcription units. From this we conclude that UBF is associated both with transcriptionally active and inactive rRNA genes and, therefore, changes in the intracellular localization of UBF are very likely not involved in rDNA transcription regulation.

Animals↗
PubMed1991

Native yeast telomeres are sufficient to stabilize linear DNA in Xenopus laevis oocytes.

We have constructed a linear plasmid in yeast containing the entire bovine papillomavirus genome and tested its physical stability following microinjection into stage VI oocytes of Xenopus laevis. Our results show that unmodified telomeres, in contrast to the yeast-passaged telomeres, drastically affect the stability of the injected linear plasmid. Plasmids carrying unmodified Tetrahymena thermophila telomeric sequences are rapidly degraded in oocytes. When these plasmids are passed through yeast, the telomere ends become modified by the addition of yeast telomeric sequences. These plasmids are stably maintained in X. laevis oocytes, demonstrating that yeast-modified telomeres are sufficient to prevent linear DNA degradation.

Animals↗
PubMed1991

Faithful in vivo transcription termination of Xenopus laevis rDNA. Correlation of electron microscopic spread preparations with S1 transcript analysis.

DNA sequencing and subsequent functional in vitro analysis of the Xenopus laevis rDNA transcription termination has led to the identification of three transcription termination sequence elements: T1, located at the 3' end of the 28S rDNA; T2, a putative processing site 235 bp downstream of T1; T3, the principal terminator positioned 215 bp upstream of the gene promoter. As demonstrated for nuclear run-off assays, T3 was found to be the main terminator for Xenopus rDNA transcription. These in vitro data are in obvious contradiction to results obtained by electron microscopic (EM) spread preparations from rapidly isolated amplified oocyte nucleoli, i.e., an rDNA chromatin probe thought to represent the in vivo situation, indicative of transcription termination at sites T1-2. However, most interestingly, T3 had--again by the EM method--been identified as the exclusive terminator for NTS spacer transcription units. In order to answer the question of whether read-through transcription of the complete rDNA spacer sequence is obligatory for 40S pre-rRNA in vivo transcription, we analyzed several hundreds of spread rRNA genes from Xenopus oocyte nucleoli in great detail, applying two different spreading procedures, e.g., dispersal of amplified oocyte nucleoli shortly in detergent-free or detergent containing low-salt media prior to the EM spreading technique. Quantitation of EM spreads resulted in the finding that read-through rDNA spacer transcription beyond T1-2 termination sites (i.e., indicative of T3 transcription termination) can be visualized for the in vivo situation at a frequency of less than 3% of rRNA genes analyzed.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1991

Working with the confocal scanning UV-laser microscope: specific DNA localization at high sensitivity and multiple-parameter fluorescence.

The use of fast-staining DNA-specific dyes such as DAPI or Hoechst 33342/33258 has been a major problem for confocal scanning laser microscopy (CSLM) studies of intranuclear chromatin organization. Moreover, the availability of a confocal ultraviolet scanning laser microscope configuration, which allows an excitation at wavelengths of 364 nm as well as 488, 514 and 543 nm, is a prerequisite for single as well as multiple fluorescence parameter studies, especially if these studies are concerned with the precise localization of intranuclear signals. Here we report the characteristics and application of a CSLM, which was adapted for UV-excitation and therefore enables comparison of the spatial distribution of several types of signals within one preparation. In addition to multiple-parameter studies, we have also investigated the sensitivity of the system with regard to the identification of the double-stranded DNA of lampbrush chromosome loops in germinal vesicles of amphibian oocytes.

Animals↗
PubMed1990

Structural organization of Acheta rDNA. Evidence for differential amplification of soma and germ-line-specific rDNA sequences.

Amplification is one of the mechanisms whereby the expression of genes can be specifically reinforced. Ribosomal gene amplification in amphibian and insect oocytes is a particularly well documented case. We studied heterogeneity, amplification and size of Acheta domesticus (insects; Orthoptera) ribosomal DNA and characteristics of male and female somatic or germ line rDNAs by analysis of genomic clones from a conventional and a microclone library. The length of the Acheta rDNA repeat unit (transcription unit and non-transcribed spacer (NTS] varied from 47 x 10(3) to 60 x 10(3) base-pairs, with highest variability within the NTS region. Deletions, fragment length heterogeneity and size variability in small steps of individual NTS segments are responsible for the observed size variation. The number of rDNA repeat units per haploid genome of Acheta was determined as 190(+/- 10%). The rDNA is amplified 14(+/- 10%)-fold in the oocyte, producing about 10,000 gene copies per cell. Our results show that the amplification mechanism does not favor individual fragments within the repeat unit. Thus, it can be concluded that amplification does not change the chromosomal characteristics of the rDNA pool. Two fragments specific for oocyte rDNA suggest that the rearrangements accompanying amplification are preferentially located in one distinct EcoRI fragment. Certain regions of Acheta rDNA contain cell-type-specific fragments: it was thus possible to characterize one purely male fragment and a second one specific for male and female soma but not for germ line rDNA. We show that Acheta rDNA reveals a combination of many features reported from different organisms and novel tissue-specific alterations on an extremely large repeat unit. The tissue-specific alterations indicate sexual and soma/germ line differentiation events that are derived by as yet unknown mechanisms.

Animals↗
PubMed1990

Plasmid and bacteriophage lambda-DNA show differential replication characteristics following injection into fertilized eggs of Xenopus laevis: dependence on period and site of injection.

The fate of DNA injected into in vitro fertilized eggs of Xenopus laevis during subsequent early embryogenesis was investigated by changing the time period and the area of injection. Form I/II plasmid DNA was found to be preferentially replicated in embryos which had been injected 60-65 min after fertilization into the animal half of the fertilized egg, irrespective of the presence of a eukaryotic origin of replication sequence element in the DNA probe used for injection. In the experiments where plasmid DNA and lambda-DNA were coinjected, only the latter was actively replicated, which suggests an inhibitory activity of this DNA on replication of coinjected plasmid DNA.

Animals↗
PubMed1990

Methodical aspects of 3-D reconstruction of chromatin architecture in mouse trophoblast giant nuclei.

Precise 3-D reconstruction of the spatial organization of murine trophoblast giant-cell chromatin is a prerequisite for detailed investigations on the fine structural changes in chromatin-fibre organization during the trophoblast endomitotic cell cycle. It appears very likely that sequential fine structural changes in the chromatin arrangement are concomitant with the changes in the gene expression pattern of these cells during the early phase of murine gestation. The complex intra-nuclear chromatin organization of mouse trophoblast giant nuclei was investigated in permanent tissue preparations which had been stained with a DNA-specific dye. The spatial chromatin arrangement was examined in fluorescence with a confocal scanning laser microscope. Serial optical sections were recorded and subjected to a computer-based 3-D reconstruction method which is suitable even for very complex biological structures.

Algorithms↗
PubMed1990

Towards light microscopic imaging of hydrated 'native' ribosomal RNA genes. A combined video microscopic and transmission electron microscopic analysis.

Investigations of the higher-order structure of 'native' genes as well as antibody localization to defined gene chromatin areas require elaborate light microscopic imaging of the chromatin structure of interest, such as visualization of unfixed and unstained gene transcription units. Since all our present structural information on the, relatively, small nucleolar rRNA genes has been obtained using transmission electron microscopy of spread genes following fixation, staining and complete dehydration, this type of EM specimen was our first specimen for LM imaging. Using Normarksi differential interference contrast video microscopy, it was found that spread EM chromatin can be sufficiently visualized by light microscopy to allow precise correlation with the conventional TEM image. In addition, it is demonstrated that video-enhanced LM can provide enough contrast enhancement for rapid visualization of spread 'native' rRNA genes in slightly fixed, fully hydrated, unstained gene chromatin.

Animals↗