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C Cremer

Publications and source records attributed to C Cremer.

At least 73 records · Page 4Linked to original sources
PubMed1989

Determination of the electrophoretic mobility of chromosomes by free flow electrophoresis. I. Morphology and stability.

Isolated metaphase chromosomes of several fibroblastoid cell lines (Chinese hamster, Chinese hamster x human hybrid) were subjected to free flow electrophoresis (FFE) to study their electrophoretic mobility (EM). The morphology and stability of the chromosomes were unaffected by FFE as examined by cytogenetic methods and flow cytometry. The chromosomes of the complement all showed similar EM under most of the conditions applied. At neutral pH the EM of the chromosomes had the same sign as free DNA and about 2/3 of its magnitude. The variation of EM with buffer parameters such as ionic strength, valence of counterions, buffer capacity and dielectric constant of the solvent were investigated. Thermal denaturation increased the EM of the chromosomes by 20%. Partial denaturation might offer a possibility to separate or enrich large amounts of chromosomes by FFE.

Animals↗
PubMed1989

Double in situ hybridization in combination with digital image analysis: a new approach to study interphase chromosome topography.

Double in situ hybridization with mercurated and biotinylated chromosome specific DNA probes in combination with digital image analysis provides a new approach to compare the distribution of homologous and nonhomologous chromosome targets within individual interphase nuclei. Here we have used two DNA probes representing tandemly repeated sequences specific for the constitutive heterochromatin of the human chromosomes 1 and 15, respectively, and studied the relative arrangements of these chromosome targets in interphase nuclei of human lymphocytes, amniotic fluid cells, and fibroblasts, cultivated in vitro. We have developed a 2D-image analysis approach which allows the rapid evaluation of large numbers of interphase nuclei. Models to test for a random versus nonrandom distribution of chromosome segments are discussed taking into account the three-dimensional origin of the evaluated 2D-distribution. In all three human diploid cell types the measurements of target-target and target-center distances in the 2D-nuclear image revealed that the labeled segments of the two chromosomes 15 were distributed both significantly closer to each other and closer to the center of the nuclear image than the labeled chromosome 1 segments. This result can be explained by the association of nucleolus organizer regions on the short arm of chromosome 15 with nucleoli located more centrally in these nuclei and does not provide evidence for a homologous association per se. In contrast, evaluation of the interphase positioning of the two chromosome 1 segments fits the random expectation in amniotic fluid and fibroblast cells, while in experiments using lymphocytes a slight excess of larger distances between these homologous targets was occasionally observed. 2D-distances between the labeled chromosome 1 and 15 segments showed a large variability in their relative positioning. In conclusion our data do not support the idea of a strict and permanent association of these homologous and nonhomologous targets in the cell types studied so far.

Cell Nucleolus↗
PubMed1989

[Regulatory aspects of the toxicological records of food additives].

An important effort is actually paid by the Commission of the European Communities in order to standardize the national legislations dealing with the food additives. Up to now, however, only recommendations have been proposed and their implementation will probably need some more years. In the mean time the Belgian Legislator has clearly defined the rules to be followed for inscription of new food additives as well as for changes of content and any other condition of authorization. The request should include a series of administrative informations as well as technical details on the characteristics of the food additive proposed for commercialization.

Belgium↗
PubMed1988

Sorting of chromosomes by magnetic separation.

Chromosomes were isolated from Chinese hamster x human hybrid cell lines containing four and nine human chromosomes. Human genomic DNA was biotinylated by nick translation and used to label the human chromosomes by in situ hybridization in suspension. Streptavidin was covalently coupled to the surface of magnetic beads and these were incubated with the hybridized chromosomes. The human chromosomes were bound to the magnetic beads through the strong biotin-streptavidin complex and then rapidly separated from nonlabeled Chinese hamster chromosomes by a simple permanent magnet. The hybridization was visualized by additional binding of avidin-FITC (fluorescein) to the unoccupied biotinylated human DNA bound to the human chromosomes. After magnetic separation, up to 98% of the individual chromosomes attached to magnetic beads were classified as human chromosomes by fluorescence microscopy.

Animals↗
PubMed1987

A method for nucleic acid hybridization to isolated chromosomes in suspension.

A procedure was developed to provide differential fluorescent staining of metaphase chromosomes in suspension following nucleic acid hybridization. For this purpose metaphase chromosomes were isolated from a Chinese hamster X human hybrid cell line. After hybridization with biotinylated human genomic DNA, the human chromosomes were visualized by indirect immunofluorescence using antibodies against biotin and fluoresceine-isothiocyanate-(FITC)-labeled second antibodies. This resulted in green fluorescent human chromosomes. In contrast, Chinese hamster chromosomes revealed red fluorescent staining only when counterstained with propidium iodide. Notably, interspecies chromosomal rearrangements could be easily detected. After hybridization and fluorescent staining, chromosomes still showed a well-preserved morphology under the light microscope. We suggest that this procedure may have a useful application in flow cytometry and sorting.

Animals↗
PubMed1986

Induction of chromosome shattering by ultraviolet light and caffeine: the influence of different distributions of photolesions.

Cells of synchronized and of asynchronously growing cultures of a V79 Chinese hamster line were microirradiated with a low power laser-UV-microbeam of wavelength 257 nm. Ultraviolet light was either focused onto a small part of the nucleus (mode I) or distributed over the whole nucleus (mode II). Following microirradiation, the cells were incubated for 7-20 h with caffeine (1-2 mM) until chromosome preparation was performed. After both modes of microirradiation, shattering of the entire chromosome complement (generalized chromosome shattering, GCS) was observed. It is suggested that the probability by which GCS is induced depends on the total number of DNA lesions rather than on their distribution in the chromatin. The results are consistent with the prediction of a "factor depletion model" which assumes that in a given cell, GCS takes place both in irradiated and non-irradiated chromosomes if the total number of daughter strand-repair sites surpasses a threshold value.

Animals↗
PubMed1984

Preparative dual-beam sorting of the human Y chromosome and in situ hybridization of cloned DNA probes.

Bivariate Hoechst/chromomycin flow karyotypes for chromosomes from a Chinese hamster-human hybrid cell line (CH-Y-VII) were established that have retained a human Y chromosome. These bivariate flow karyotypes showed the human Y chromosome to be completely separated from the peaks for the Chinese hamster chromosomes. In preparative dual-beam sorting experiments, 3 X 10(6) chromosomes were sorted from the Y peak into frozen petri dishes. An examination of Q-banded samples of sorted chromosomes revealed that 82% +/- 5% of them were human Y chromosomes. The DNA from the sorted chromosomes (approximately 250 ng) was isolated and used to establish a genomic library (vector lambda gt WES. lambda B). Three clones (YACG 45, 52, 54) of this library containing inserts of repetitive human DNA were used for chromosomal localization by means of in situ hybridization to metaphase spreads of male human lymphocytes and of CH-Y-VII cells. In all three cases, a significant binding to the human Y chromosome was observed. A more detailed study of the chromosomal distribution of sequences homologous to the insert of YACG 45 suggested the existence of minor binding sites on several human autosomes. Southern blot analysis revealed the existence of other human specific sequences without Y chromosome specificity.

Animals↗
PubMed1984

Sister chromatid exchange (SCE) induced by laser-UV-microirradiation: correlation between the distribution of photolesions and the distribution of SCEs.

Small, medium, and large nuclear areas comprising approximately 5, 30, or 80% of the total area of the interphase nuclei of Chinese hamster cells (M3-1) cultivated in vitro were irradiated with a laser-UV-microbeam of wavelength 257 nm. The DNA of the cells was substituted with 5-bromodeoxyuridine (BrdUrd) for 1 cell cycle in one set of experiments. After microirradiation the cells were grown for a second cycle in medium without BrdUrd (protocol A). In a second set, cells with nonsubstituted DNA were microirradiated and grown for 2 additional cycles, the first in the presence, the second in the absence of BrdUrd (protocol B). In situ chromosome preparation and differential chromatid staining was subsequently performed. The induction of sister chromatid exchanges (SCEs) was found to be dependent on both the ultraviolet (UV) dose and the spatial distribution of the UV energy within the cell nucleus. Following both protocols the average number of chromosomes with SCEs was significantly higher after microirradiation of a large nuclear area as compared to microirradiation of a small nuclear area. In the latter case, multiple SCEs were noted on individual chromosome arms at the first postirradiation mitosis (protocol A). In other cells, especially at higher doses, protocol A resulted in shattering of a few closely neighbored chromosomes which were surrounded by intact ones with normal SCE levels. Microirradiation of medium-sized nuclear areas produced high levels of SCEs over a number of chromosomes which still appeared spatially related in a part of the metaphase spread. Finally, high SCE levels could be observed over most or all chromosomes when a large nuclear area (up to 100%) was exposed to the microbeam. Following protocol B the increase of SCEs was much less pronounced. Microirradiation of a small part of the cytoplasm in addition to the nuclei did not induce SCEs. Our results support the concept (i) that interphase chromosomes occupy distinct nuclear domains and indicate (ii) that the induction of SCEs by UV light is restricted to microirradiated chromatin.

Animals↗
PubMed1984

Chromosome assignment of two cloned DNA probes hybridizing predominantly to human sex chromosomes.

In situ hybridization experiments were carried out with two clones, YACG 35 and 2.8, which had been selected from two genomic libraries strongly enriched for the human Y chromosome. Besides the human Y chromosome, both sequences strongly hybridized to the human X chromosome, with few minor binding sites on autosomes. In particular, on the X chromosome DNA from clone YACG 35 hybridized to the centromeric region and the distal part of the short arm (Xp2.2). On the Y chromosome, the sequence was assigned to one site situated in the border region between Yq1.1 and Yq1.2. DNA from clone 2.8 also hybridized to the centromeric region of the X and the distal part of the short arm (Xp2.2). On the Y, however, two binding sites were observed (Yp1.1 and Yq1.2). The findings indicate that sex chromosomal sequences may be localized in homologous regions (as suggested from meiotic pairing) but also at ectopic sites.

Animals↗
PubMed1983

Cloning of genomic sequences from the human Y chromosome after purification by dual beam flow sorting.

Human Y chromosomes were purified by dual beam flow sorting from a human X Chinese hamster cell line retaining the Y as the only free human chromosome. DNA was extracted from the Y fraction and cloned into lambda gtWES . lambda B vector arms. More than 100 recombinant clones carrying human inserts have been characterised by Benton-Davis plaque screening and Southern blotting or in situ hybridisation. Several repetitive sequences were found to be predominantly located on the Y, whereas the majority also cross-hybridised with autosomal DNA. One repetitive clone gave a specific hybridisation signal with the X and the Y chromosome but not with autosomes. Preliminary evidence indicates that many clones contain single copy as well as repetitive sequences. However, no Y-specific single copy sequence has yet been identified.

Animals↗
PubMed1983

Immunocytochemical localization of chromatin regions UV-microirradiated in S phase or anaphase. Evidence for a territorial organization of chromosomes during cell cycle of cultured Chinese hamster cells.

Chinese hamster cells (M3-1 line) in S phase were laser-UV-microirradiated (lambda, 257 nm) at a small site of the nucleus. Cells were fixed either immediately thereafter or in subsequent stages of the cell cycle, including prophase and metaphase. The microirradiated chromatin was visualized by indirect immunofluorescence microscopy using antibodies specific for UV-irradiated DNA. During the whole post-incubation period (4-15 h) immunofluorescent labelling was restricted to a small part of the nucleus (means, 4.5% of the total nuclear area). In mitotic cells segments of a few chromosomes only were labelled. Following microirradiation of chromosome segments in anaphase, immunofluorescent labelling was observed over a small part of the resulting interphase nucleus. A territorial organization of interphase chromosomes, i.e. interphase chromosomes occupying distinct domains, has previously been demonstrated by our group for the nucleus of Chinese hamster cells in G1. Our present findings provide evidence that this organization pattern is maintained during the entire cell cycle.

Anaphase↗
PubMed1983

UV micro-irradiation of the Chinese hamster cell nucleus and caffeine post-treatment. Immunocytochemical localization of DNA photolesions in cells with partial and generalized chromosome shattering.

UV micro-irradiation of a small part of the Chinese hamster nucleus and caffeine post-incubation often results in shattered chromosomes at the first post-irradiation mitosis. In some of these mitotic cells, chromosome shattering is restricted to a few chromosomes spatially related in a small area of the metaphase spread; in others, shattering includes the whole chromosome complement. These 2 types of damage have been called partial and generalized chromosome shattering (PCS and GCS). Using antisera that specifically react with UV-irradiated DNA, we identified micro-irradiated chromatin in interphase nuclei and in mitotic cells with PCS or GCS by indirect immunofluorescence microscopy. In PCS, immunofluorescence staining was found in the damaged area, while the surrounding intact chromosomes were not stained. In GCS, staining was also restricted to a small region of the shattered chromosome complement. In other experiments, cells synchronized in G1 were micro-irradiated in the nucleus, pulse-labelled with [3H]thymidine and post-incubated with caffeine. Autoradiographs of cells with GCS showed unscheduled DNA synthesis restricted to a small chromatin region. Our data present direct evidence that the distribution of DNA photolesions does not coincide with the sites of chromosomal damage in GCS. As a working, hypothesis, we propose that an indirect mechanism is involved in the induction of GCS by which DNA photolesions in a small nuclear segment induce shattering of both micro-irradiated and non-irradiated chromosomes.

Animals↗
PubMed1982

Induction of chromosome damage by ultraviolet light and caffeine: correlation of cytogenetic evaluation and flow karyotype.

Asynchronously growing cells of a M3-1 Chinese hamster line were ultraviolet (UV) irradiated (lambda = 254 nm) with UV fluences up to 7.5 J/m(2). After irradiation cells were incubated with or without 2 mM caffeine for 20 hr, then mitotic cells were selected by mechanical shaking. Their chromosomes were isolated, stained with Hoechst 33258 and chromomycin A3, and measured flow cytometrically. While the fluorescence distributions of chromosomes (flow karyo-types) from cells treated with UV alone or with caffeine alone were very similar to those of untreated controls, the flow karyo-types of UV + caffeine-treated cells showed a debris continuum that increased with increasing UV fluence suggesting an increased number of chromosome fragments. Visual evaluation of metaphase plates revealed that the percentage of cells with chromosome damage also increased steadily with increasing UV fluence. A high degree of correlation was observed between the relative magnitude of the debris level from flow karyotypes and the percentage of cells with chromosome damage and with generalized chromosomes shattering, respectively, as determined from metaphase spreads.

Animals↗
PubMed1982

Flow cytometric characterization of a Chinese hamster X man hybrid cell line retaining the human Y chromosome.

A Chinese hamster X man hybrid cell line (CH-Y-VII) was established which retains a free human Y chromosome. Exponentially growing CH-Y-VII cells were arrested with colcemid; metaphase chromosomes were isolated and stained with 33258 Hoechst (HO) plus Chromomycin A3 (CA3), or with ethidium bromide (EB). The HO/CA3-stained chromosomes were measured in a dual beam flow cytometer, and bivariate HO/CA3 flow karyotypes and univariate HO and CA3 flow karyotypes were established. EB-stained chromosomes were analyzed in a modified Becton Dickinson FACS-Sorter. For all three stains used, the human Y chromosome forms a separate peak in univariate flow karyotypes; the optimum resolution was obtained for the HO distribution. In the bivariate HO/CA3 flow karyotype, the peak for the human Y chromosome is completely separated from the Chinese hamster chromosomes.

Animals↗