Biomedical subjects

C Cremer

Publications and source records attributed to C Cremer.

100 records · Page 6Linked to original sources

PubMedDate not supplied

Preparative continuous separation of biological particles by means of free-flow magnetophoresis in a free-flow electrophoresis chamber.

For sorting, cells or cellular components can specifically be labeled by antibody-coated magnetic beads. We have developed a device for continuous magnetic sorting based on the flow-chamber of a free-flow electrophoresis system. Magnetically labeled particles are injected into a given continuously flowing chamber buffer and pass an inhomogeneous magnetic field, configurated perpendicular to the flow direction. According to its magnetic moment, the magnetic material is deviated into the direction of the magnetic forces, while nonmagnetic material passes the field without interaction. The magnetic forces can be changed with the electrical current of the solenoids producing the magnetic field. As in the free-flow electrophoresis system, the particle fractions are collected in different vials. On-line control of the experiments can be performed by an optical scanning system. Experiments with model particles achieved a sorting purity of more than 99% at a rate of up to 5 X 10(8) particles per hour. In experiments with blood cells, a high enrichment of either B-or-T-lymphocytes was obtained. In contrast to free-flow electrophoresis, there is no limitation, in principle, regarding the type of chamber buffer to be used. This allows an optimal adaptation of the buffer conditions to the requirements of vital sorting. The preliminary results so far confirm this conclusion.

B-Lymphocytes↗

PubMedDate not supplied

Slit scan flow cytometry of isolated chromosomes following fluorescence hybridization: an approach of online screening for specific chromosomes and chromosome translocations.

The recently developed methods of non radioactive in situ hybridization of chromosomes offer new aspects for chromosome analysis. Fluorescent labelling of hybridized chromosomes or chromosomal subregions allows to facilitate considerably the detection of specific chromosomal abnormalities. For many biomedical applications (e.g. biological dosimetry in the low dose range), a fast scoring for aberrations (e.g. dicentrics or translocations) in required. Here, we present an approach depending on fluorescence in situ hybridization of isolated suspension chromosomes that indicates the feasibility of a rapid screening for specific chromosomes or translocations by slit scan flow cytometry. Chromosomes of a Chinese hamster x human hybrid cell line were hybridized in suspension with biotinylated human genomic DNA. This DNA was decorated with FITC by a double antibody system against biotin. For flow cytometry the chromosomes were stabilized with ethanol and counterstained with DAPI or propidium iodide (PI). An experimental data set of several hundred double profiles was obtained by two parameter slit scan flow cytometry and evaluated automatically. The evaluation algorithm developed allowed a classification of chromosomes according to the number of centromeres and their chromosomal positions in less than 1 msec per individual profile. Approximately 20% of the measured DAPI profiles showed a bimodal distribution with a significant centromeric dip indicating a "normal" chromosomal morphology and a correct alignment in the flow system. In many cases, profiles of a "normal" bimodal fluorescence distribution of the DNA stain (DAPI, PI) were correlated with a "normal" FITC profile. Due to their centromeric indices these profiles agreed well to the expected human chromosomes of the cell line. In some cases of "normal" DAPI (PI) profiles, "aberrant" FITC profiles were observed.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMedDate not supplied

A simplified combination of DNA probe preparation and fluorescence in situ hybridization.

Fluorescence in situ hybridization (FISH) has found widespread applications in cytogenetics. So far the standard protocols for probe amplification (and simultaneous labeling) by PCR, nick translation and in situ hybridization involve different buffer systems leading to a number of time consuming washing steps even before hybridization. In this manuscript we show a fast technique of a close combination of DNA probe preparation and in situ hybridization (ISH). This method was applied to metaphase chromosomes from human lymphocytes fixed on slides. Two specific repetitive DNA probes, the pUC 1.77 DNA probe and the DYZ1 repetitive DNA fraction were used, amplified and labeled in different ways. Additional experiments with total genomic male human DNA as the DNA probe suggest that this method may be extended to a large variety of other probes. Moreover the ISH technique described does not require toxic denaturing agents, such as formamide.

Chromosomes, Human↗

PubMedDate not supplied

Identification and cytogenetic analysis of an abnormal pig chromosome for flow cytometry and sorting.

For cytogenetics of pig (Sus scrofa domestica) and the influence of chromosome aberrations on pig production, high interest exists in flow sorted chromosomes for gene mapping, to establish DNA-libraries, or to produce DNA-probes. Flow karyotyping and sorting as well as slit scan flow analysis of metaphase chromosomes of an abnormal cell type carrying a translocation marker chromosome 6/15 are described. Flow sorting of the largest chromosomes of these cells was performed. After sorting the chromosomes still had a well preserved morphology and were identified microscopically by G-banding. The quality of the band pattern of the sorted chromosomes was compatible to that of isolated chromosomes not subjected to flow cytometry. The sorted fraction showed an enrichment of chromosome 6/15 and chromosome 1 which have quantitatively about the same integrated fluorescence intensity. Slit scan flow analysis was performed to discriminate these two chromosomes. Metacentric and submetacentric chromosomes were analyzed according to their bimodal slit scan profiles. Profiles of the largest chromosomes were distinguished by their different centromeric indices. Two groups were interpreted as the normal chromosome 1 and the translocation chromosome 6/15.

Animals↗

PubMedDate not supplied

Optimization of fast-fluorescence in situ hybridization with repetitive alpha-satellite probes.

A rapid FISH (fluorescence in situ hybridization) technique (Fast-FISH) for quantitative microscopy has been recently introduced. For highly repetitive DNA probes the hybridization (renaturation) time and the number of necessary washing steps were reduced considerably by omitting formamide or equivalent denaturing chemical agents. Due to low stringency conditions major and minor binding sites of the probes used showed visible FISH signals well suited for quantitative image-microscopy. The discrimination of minor and major binding sites was possible by automated image-processing. Here, a further, quantitative optimization of the Fast-FISH technique is described that allows to clearly discriminate major and minor binding sites of alpha-satellite probes by an easy image classification parameter. With respect to the optimization it was necessary to verify two sensitive parameters (hybridization time and temperature) of the given rapid FISH protocol. As examples the systematic optimization for the two probes D12Z2 (major binding site on the centromere of chromosome 12) and D8Z2 (major binding site on the centromere of chromosome 8) are shown. The optimal hybridization conditions concerning rapidness and quality of chromosome morphology were obtained using a hybridization temperature of 70 degrees C and a hybridization time of 60 min. For these conditions major and minor binding sites were clearly discriminated by the intensity maximum Smax of the corresponding FISH-spots.

Binding Sites↗

PubMedDate not supplied

Painting of human chromosome 8 in fifteen minutes.

The technique of chromosome-in-situ suppression (CISS)-hybridization (chromosome painting) has now been well established. However, all standard protocols so far require long renaturation times (typically 12 hours and more). Here, we describe a new, extremely fast protocol for chromosome painting using a commercially available, directly fluorescence labelled probe for chromosome 8. The hybridization conditions used omit separate preannealing procedures and denaturing chemical agents. The renaturation time required for chromosome painting was reduced to 15 minutes. In addition, most washing steps were eliminated. As a consequence, the entire painting procedure was feasible in less than half an hour.

Chromosomes, Human, Pair 8↗

PubMedDate not supplied

The influence of formamide on thermal denaturation profiles of DNA and metaphase chromosomes in suspension.

Systematic photometric studies are presented to analyze the thermal denaturation behaviour with and without formamide of metaphase chromosome suspensions in comparison to DNA solutions. Temperature dependent hyperchromicity measurements at 256 nm and 313 nm were performed using an appropriately designed computer-controlled photometer device. Due to an upright optical axis, this allowed absorbance measurements with negligible sedimentation effects not only for solutions of pure DNA, but also for particle suspensions of isolated metaphase chromosomes. This device has a temperature resolution of +/- 0.5 degrees C and an optical sensitivity of 10(-3) to 10(-4) optical density. For calf thymus DNA the reduction of the melting point with the increase of formamide in the solution was measured at pH 7.0 and pH 3.2. The good correlation of the theoretical approximation to experimental data indicated the suitability of the apparatus to quantitatively describe DNA conformation changes induced by thermal denaturation. For metaphase chromosome preparations of Chinese hamster culture cells, absorbance changes were measured between 20 degrees C and 95 degrees C with a temperature gradient of 1 degrees C/min. These measurements were performed at pH 7.0 and at pH 3.2. The denaturation profiles (= first derivative of the absorbance curve) resulted in a highly variable peak pattern at 256 nm and 313 nm indicating complex conformation changes. A statistical evaluation of the temperature values of the peak maxima resulted in temperature ranges typical for chromosomal conformation changes during thermal treatment. Especially the range of highest temperature values was independent from pH modifications. For pH 3.2 the influence of formamide on the denaturation behaviour of metaphase chromosome preparations was analyzed. In contrast to pure DNA solutions, a reduction of the "melting point" (i.e. the maximum temperature at which a conformation change takes place) was not found. However, the denaturation behaviour depended on the duration of formamide treatment before the measurement.

Animals↗

PubMedDate not supplied

Rise, fall and resurrection of chromosome territories: a historical perspective. Part I. The rise of chromosome territories.

It is now generally accepted that chromosomes in the cell nucleus are organized in distinct domains, first called chromosome territories in 1909 by the great cytologist Theodor Boveri. Yet, even today chromosomes have remained enigmatic individuals, whose structures, arrangements and functions in cycling and post-mitotic cells still need to be explored in full detail. Whereas numerous recent reviews describe present evidence for a dynamic architecture of chromosome territories and discuss the potential significance within the functional compartmentalization of the nucleus, a comprehensive historical account of this important concept of nuclear organization was lacking so far. Here, we describe the early rise of chromosome territories within the context of the discovery of chromosomes and their fundamental role in heredity, covering a period from the 1870th to the early 20th century (part I, this volume). In part II (next volume) we review the abandonment of the chromosome territory concept during the 1950th to 1980th and the compelling evidence, which led to its resurrection during the 1970th to 1980th.

Animals↗

PubMedDate not supplied

Rise, fall and resurrection of chromosome territories: a historical perspective. Part II. Fall and resurrection of chromosome territories during the 1950s to 1980s. Part III. Chromosome territories and the functional nuclear architecture: experiments and models from the 1990s to the present.

Part II of this historical review on the progress of nuclear architecture studies points out why the original hypothesis of chromosome territories from Carl Rabl and Theodor Boveri (described in part I) was abandoned during the 1950s and finally proven by compelling evidence forwarded by laser-uv-microbeam studies and in situ hybridization experiments. Part II also includes a section on the development of advanced light microscopic techniques breaking the classical Abbe limit written for readers with little knowledge about the present state of the theory of light microscopic resolution. These developments have made it possible to perform 3D distance measurements between genes or other specifically stained, nuclear structures with high precision at the nanometer scale. Moreover, it has become possible to record full images from fluorescent structures and perform quantitative measurements of their shapes and volumes at a level of resolution that until recently could only be achieved by electron microscopy. In part III we review the development of experiments and models of nuclear architecture since the 1990s. Emphasis is laid on the still strongly conflicting views about the basic principles of higher order chromatin organization. A concluding section explains what needs to be done to resolve these conflicts and to come closer to the final goal of all studies of the nuclear architecture, namely to understand the implications of nuclear architecture for nuclear functions.

Animals↗

PubMedDate not supplied

Non-enzymatic, low temperature fluorescence in situ hybridization of human chromosomes with a repetitive alpha-satellite probe.

In all DNA-DNA in situ hybridization (ISH) procedures described so far in the literature, the production of single-stranded target DNA sequences plays a decisive role. This can be achieved either by enzymatic treatment at physiological temperatures or by the separation of double-stranded DNA sequences. Denaturation by heat and chemical agents (e.g. formamide) is regarded as a prerequisite for the non-enzymatic ISH process. However, additional mechanisms of a non-enzymatic ISH procedure are conceivable which do not require high temperature treatment combined with formamide. Here, we report on a non-enzymatic, non-formamide, low temperature, fluorescence, in situ hybridization (FISH) procedure which allowed a microscopic visualization and quantitative fluorescence analysis of the binding sites of a repetitive DNA probe. Following only probe denaturation at 94 degrees C, hybridization was performed at 52 degrees C for 30 min, i.e. at nearly physiological temperatures. Moreover, increasing the hybridization time to 3 hours indicated that hybridization sites became also visible at 37 degrees C. Since the protocols are based on recently described Fast FISH developments, the technique will be called Low Temperature Fast-FISH (LTFF).

Cell Nucleus↗