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M F Trendelenburg

Publications and source records attributed to M F Trendelenburg.

At least 37 records · Page 2Linked to original sources
PubMed1989

Transcriptional characteristics of in vitro assembled chromatin assayed by microinjection into Xenopus laevis oocytes.

Plasmid DNA was in vitro assembled into chromatin using an S-150 extract of Xenopus laevis oocytes. By varying the assembly temperature and DNA concentration it is possible to generate fully or partially assembled molecules. The fate of the in vitro preassembled molecules injected into X. laevis oocyte nuclei and their transcriptional activity were studied. Completely reconstituted molecules underwent a rearrangement of their chromatin structure after injection and showed reduced transcriptional activity compared to protein-free DNA or partially reconstituted chromatin.

Animals↗
PubMed1989

Restriction analysis of lambda EMBL3 background recombinants: occurrence of lambda phages carrying a head to tail oriented left arm DNA sequence.

Eight representative recombinant background clones of lambda EMBL3 were analysed using KpnI, BamHI, SalI, EcoRI and HindIII digestion. We found that lambda EMBL3 carries its own left arm in the BamHI cloning site. In the way, recombinant molecules were found to be generated which can grow on Escherichia coli strain NM539. In all cases analysed, the left arm DNA was inserted in a head to tail orientation. Seven clones carried a restored BamHI site at the cos site-BamHI site connection. In the region where the inserted left arm and the right arm were ligated, BamHI cloning produces a large palindromic sequence consisting of two polylinkers. This BamHI site was incompletely cleaved in all cases analysed. We assume that a part of the lambda DNA molecule in this region shows a cruciform structure prohibiting recognition or cleavage of this site by restriction endonuclease BamHI.

Bacteriophage lambda↗
PubMed1989

Expression of mouse histone H1(0) promoter sequences following microinjection into Xenopus oocytes and developing embryos.

Evidence from expression studies using transfected F9 teratocarcinoma stem cells indicates that the synthesis of the H1(0) histone is turned on very soon after the cells have been treated with retinoic acid, which causes them to differentiate into murine parietal endoderm. This increase in H1(0) at the time of commitment would allow a reorganization of the chromatin with a reprogramming of the gene activity of undifferentiated F9 cells to the differentiated state. The particular interest of the further development of this differentiation model is to answer the question whether this specific stimulation of expression can be induced only in homologous differentiation systems, or whether the identified H1(0) promoter sequences can also be specifically stimulated by heterologous factors. We therefore injected the mammalian H1(0) promoter sequences into Xenopus oocytes and fertilized eggs. The results of oocyte injection experiments indicate that H1(0) promoter sequence elements similar to those used in transfected F9 cells are specifically expressed in the oocyte. For analysis of H1(0) expression in Xenopus embryos we used promoter constructs ligated to beta-galactosidase sequences for microinjection. This procedure allows a particularly rapid and complete detection of expressed promoter clones within the differentiated tissues of the early Xenopus embryo.

Animals↗
PubMed1988

Different regulatory elements are required for cell-type and stage specific expression of the Xenopus laevis skeletal muscle actin gene upon injection in X.laevis oocytes and embryos.

In the present study, we demonstrate by transcript mapping that the injected Xenopus skeletal muscle alpha-actin gene is transcribed and spliced in Xenopus oocytes but not correctly initiated at the alpha-actin promoter. This leads to correctly spliced transcripts even if constructs without putative promoter sequences are injected. On the other hand, alpha-actin transcripts are translated in injected oocytes as shown by the detection of alpha-actin protein. By contrast, correctly initiated alpha-actin transcripts can be found in neurula embryos when the injected clone contains 5' flanking sequences extending from +27 to -680. alpha-actin gene fragments without the 680 nucleotides 5' flanking region are activated unspecifically after midblastula transition, whereas the clones carrying this region are activated correctly at the end of gastrulation. Cell type specific expression seems to be modulated by sequences within the transcribed region.

Actins↗
PubMed1988

Structural analysis of the mitotic cycle in pre-gastrula Xenopus embryos.

The long-known phenomenon of karyomere (chromosome vesicle) formation at early telophase of the nuclear cycle during early embryogenesis of a wide range of organisms including amphibians (Rubaschkin 1905; for review, see Richards 1917) was investigated in the early cleavage cycles of Xenopus laevis embryos before the mid blastula transition. Embryos were fixed and Epon embedded at successive time intervals and consecutive thick (3 micron) and ultrathin sections cut. Using conventional light microscopy at low magnification as well as phase and/or interference contrast video microscopy at high magnification, a substantial amount of information could be obtained from the analysis of optical sections in thick-sectioned material. In addition, details of the ultrastructural organization could be analysed from corresponding ultrathin sections by electron microscopy. The light microscopic analysis of serial thick sections allowed precise determination of the arrangement and sizes of telophase karyomere structures during the embryonic nuclear division cycle. It was found that small, widely spaced 1st order karyomeres fuse to larger (2nd order) karyomeres which then progressively exhibit lateral fusion of neighbouring karyomeres. The final coalescence of adjacent karyomeres marks the onset of the reorganization of the typical interphase nuclear structure. The data are discussed with regard to the occurrence of karyomeres during the embryonic nuclear cycle of arthropods, dipteran insects, and echinoderms as well as recent progress in the use of Xenopus egg extracts for in vitro assembly of nuclear structures around protein-free DNA.

Animals↗
PubMed1988

DNA-fluorescence of mammalian intra-nucleolar chromatin detected by confocal laser scanning microscopy (CLSM).

The complex spatial DNA distribution in the mammalian interphase nucleus was investigated in Feulgen stained thick sections through mouse trophoblast giant nuclei after Lowicryl embedding. DNA-fluorescence was visualized using confocal laser scanning microscopy. Our results show that the spatial arrangement of major interphase chromatin areas can be precisely documented, including the distribution of small intra-nucleolar chromatin zones.

Animals↗
PubMed1988

Video microscopic image processing facilitates the evaluation of light microscopic autoradiography at high magnification.

Light microscopic autoradiographs of 3H-thymidine labelled unstained semithin sections of Xenopus laevis embryonic nuclei were examined with conventional Nomarski differential interference contrast, phase-contrast and video microscopy. Whereas at low magnification it was possible to obtain a photograph of the nuclear structure and the silver grains in one focal plain, at high magnification, with small depths of focus, a satisfactory image was not attainable. Therefore, we stored the images of the two different focus levels with a digital image processing system and combined both images by an arithmetic operation. This video microscopic technique allows the use of high magnification light microscopy with oil immersion objectives and the application of additional electronic contrast enhancing methods for an adequate and rapid analysis of light microscopic autoradiographs.

Animals↗
PubMed1985

Structural organization of an active, chromosomal nucleolar organizer region (NOR) identified by light microscopy, and subsequent TEM and STEM electron microscopy.

The three-dimensional arrangement of the chromatin components within the nucleolar organizer regions (NORs) from living oocyte nuclei was investigated. As a suitable cell system we chose vitellogenic oocytes of the orthopteran insect Acheta. This cell type is particularly attractive for analysis of nucleolar chromatin, since structural and functional aspects of NORs during early oogenic stages (including the association of NORs with amplified rDNA copies) are particularly well known (Lima-de-Faria 1974). In the course of the present study we first identified putative chromosomal NORs in isolated nuclei of mid-diplotene oocytes according to morphological characteristics using differential interference contrast (DIC) or phase contrast light microscopy. The presence of actively transcribing chromosomal NORs during this late stage of Acheta oogenesis obviously had been overlooked by previous investigators, probably due to difficulties of chromosome visualization. For a more detailed ultrastructural analysis, NORs were gently sedimented from opened nuclei and processed for sectioning using a modified "end-embedding" procedure (Mott and Callan 1975; Spring and Franke 1981). A small number of thick and thin sections could be made from individual NORs. Sections were analyzed by light microscopy, conventional transmission electron microscopy (TEM) and scanning transmission electron microscopy (STEM). Whereas the structural connection of NORs to the chromosome axis and also the general arrangement of active nucleolar genes within the NOR complex could be seen with TEM, the visualization of individual nucleolar genes and the organization of transcription complexes was only possible using bright field STEM of thick sections at low temperature.

Animals↗
PubMed1983

Progress in visualization of eukaryotic gene transcription.

Methods for visualization of the ultrastructure of transcriptionally active eukaryotic genes have been developed using chromatin from giant nuclei of amphibian oocytes (Miller and Beatty 1969). Rapidly isolated chromatin is subjected to low salt treatment in order to dissociate most chromatin associated proteins. As a result, gene-chromatin with associated RNA polymerase particles and RNA transcripts can be directly analysed in electron microscope chromatin spread preparations. More recently, progress has been made in utilising living amphibian oocyte nuclei as a transcription system for cloned eukaryotic genes. In this article, an account of such experiments is given, with emphasis on results and problems of chromatin and transcription organization of microinjected cloned genes. The described transcription assay system possesses important potential for investigation of gene mutations and in particular for the elucidation of molecular aspects of experimental oncology and molecular human genetics.

Amphibians↗
PubMed1982

Chromatin structure of Xenopus rDNA transcription termination sites. Evidence for a two-step process of transcription termination.

The ultrastructure of Xenopus laevis (X.l.) 40S pre-rRNA transcription termination sites was investigated by electron microscopy. Amplified nucleolar chromatin was rapidly dispersed and processed for chromatin spread preparations. This type of preparation revealed that many rDNA termination sites appeared as 50 nm long segments of chromatin axis covered by a complex of three closely spaced RNA polymerase particles. Particle (Ptl) was characterized by the association with the terminal full-length pre-rRNP fibril; particles (Pt2) and (Pt3) are located downstream from (Ptl) and appear to be devoid of transcript fibrils. This particular structural arrangement as well as sequence homology analyses of 3' adjacent spacer rDNA segments indicate that transcript release and dissociation of polymerase particles are not necessarily coupled and termination of X.l. rDNA transcription may occur as a two step process. The structural data are correlated with homologies of DNA sequences at Xenopus rDNA transcription termination regions and are discussed with respect to sequence data of 3' termination sites of rRNA genes from other species.

Animals↗
PubMed1981

A nucleolar skeleton of protein filaments demonstrated in amplified nucleoli of Xenopus laevis.

The amplified, extrachromosomal nucleoli of Xenopus oocytes contain a meshwork of approximately 4-nm-thick filaments, which are densely coiled into higher-order fibrils of diameter 30-40 nm and are resistant to treatment with high- and low-salt concentrations, nucleases (DNase I, pancreatic RNase, micrococcal nuclease), sulfhydryl agents, and various nonionic detergents. This filamentous "skeleton" has been prepared from manually isolated nuclear contents and nucleoli as well as from nucleoli isolated by fluorescence-activated particle sorting. The nucleolar skeletons are observed in light and electron microscopy and are characterized by ravels of filaments that are especially densely packed in the nucleolar cortex. DNA as well as RNA are not constituents of this structure, and precursors to ribosomal RNAs are completely removed from the extraction-resistant filaments by treatment with high-salt buffer or RNase. Fractions of isolated nucleolar skeletons show specific enrichment of an acidic major protein of 145,000 mol wt and an apparent pI value of approximately 6.15, accompanied in some preparations by various amounts of minor proteins. The demonstration of this skeletal structure in "free" extrachromosomal nucleoli excludes the problem of contaminations by nonnucleolar material such as perinucleolar heterochromatin normally encountered in studies of nucleoli from somatic cells. It is suggested that this insoluble protein filament complex forms a skeleton specific to the nucleolus proper that is different from other extraction-resistant components of the nucleus such as matrix and lamina and is involved in the spatial organization of the nucleolar chromatin and its transcriptional products.

Animals↗
PubMed1981

Patterns of transcriptional activity of nucleolar genes during progesterone-induced maturation of oocytes of Xenopus laevis.

Maturation has been induced in full grown oocytes of Xenopus laevis by incubation in progesterone, and the ultrastructure of their nucleoli has been examined by electron microscopy using the chromatin spreading technique. We show that in this species numerous extrachromosomal nucleoli maintain high levels of transcription of rDNA for up to 200 min after the application of the hormone, i.e., shortly before germinal vesicle breakdown. Transcription has been identified as normal arrays of matrix units containing densely packed transcriptional complexes. In addition to normal-sized arrays of gradients of nascent RNP fibrils, as typical of active pre-rRNA genes, a number of unusual structures are described which include situations of sparse coverage of lateral fibrils in some matrix units, indicative of reduced frequencies of initiation events. The observations are discussed in relation to the time course of nucleolar gene inactivation described in oocytes of other amphibian species, as well as in relation to inactivation of chromosomal genes characteristic of this step of meiotic prophase I.

Animals↗