Localized ultraviolet laser microbeam irradiation of early Drosophila embryos: Fate maps based on location and frequency of adult defects

Drosophila embryos were locally irradiated with a 257-nm laser microbeam during blastoderm and germ band stages. Depending on stage and beam diameter (10–30 μm), from 0 to 45 nuclei were exposed to the uv radiation. The doses used, 5 or 10 erg, did not eliminate nuclei or cells at once, but up to 50% of the adult survivors from irradiated eggs carried defects in the thorax. These were scored with reference to the imaginal discs from which the affected structures derive. For each thoracic disc a “target center” was calculated as the weighted mean value of all beam locations affecting the respective adult derivatives. The target centers for the germ band stage map within the respective germ band segments. The pattern of target centers for the blastoderm stage is comparable to the thoracic region of published fate maps, and the distances between adjacent leg centers (approximately three cell diameters) agree with recent evidence based on mosaic flies. We discuss the question whether the target centers mark the position of the respective disc progenitor cells at the stages of irradiation and conclude that these positions are rendered rather correctly at least with reference to the longitudinal egg axis

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

Branching Instabilities in Rapid Fracture: Dynamics and Geometry

We propose a theoretical model for branching instabilities in 2-dimensional fracture, offering predictions for when crack branching occurs, how multiple cracks develop, and what is the geometry of multiple branches. The model is based on equations of motion for crack tips which depend only on the time dependent stress intensity factors. The latter are obtained by invoking an approximate relation between static and dynamic stress intensity factors, together with an essentially exact calculation of the static ones. The results of this model are in good agreement with a sizeable quantity of experimental data.Comment: 9 pages, 11 figure

Research in the effective implementation of guidance computers with large scale arrays Interim report

Functional logic character implementation in breadboard design of NASA modular compute

Detection of chromosome aberrations in the human interphase nucleus by visualization of specific target DNAs with radioactive and non-radioactive in situ hybridization techniques: diagnosis of trisomy 18 with probe L1.84

The localization of chromosome 18 in human interphase nuclei is demonstrated by use of radioactive and nonradioactive in situ hybridization techniques with a DNA clone designated L1.84. This clone represents a distinct subpopulation of the repetitive human alphoid DNA family, located in the centric region of chromosome 18. Under stringent hybridization conditions hybridization of L1.84 is restricted to chromosome 18 and reflects the number of these chromosomes present in the nuclei, namely, two in normal diploid human cells and three in nuclei from cells with trisomy 18. Under conditions of low stringency, cross-hybridization with other subpopulations of the alphoid DNA family occurs in the centromeric regions of the whole chromosome complement, and numerous hybridization sites are detected over interphase nuclei. Detection of chromosome-specific target DNAs by non-radioactive in situ hybridization with appropriate DNA probes cloned from individual chromosomal subregions presents a rapid means of identifying directly numerical or even structural chromosome aberrations in the interphase nucleus. Present limitations and future applications of interphase cytogenetics are discussed

Differences of size and shape of active and inactive X-chromosome domains in human amniotic fluid cell nuclei

It is a widely held belief that the inactive X-chromosome (Xi) in female cell nuclei is strongly condensed as compared to the largely decondensed active X-chromosome (Xa). We have reconsidered this problem and painted X-chromosome domains in nuclei of subconfluent, female and male human amniotic fluid cell cultures (46, XX and 46, XY) by chromosomal in situ suppression (CISS) hybridization with biotinylated human X-chromosome specific library DNA. FITC-conjugated avidin was used for probe detection and nuclei were counterstained with propidium iodide (PI). The shape of these nuclei resembling flat ellipsoids or elliptical cylinders makes them suitable for both two-dimensional (2D) and three-dimensional (3D) analyses. 2D analyses of Xi- and Xa-domains were performed in 34 female cell nuclei by outlining of the painted domains using a camera lucida. Identification of the sex chromatin body in DAPI-stained nuclei prior to CISS-hybridization was confirmed by its colocalization with one of the two painted X-domains. In 31 of the 34 nuclei the area AXi for the inactive X-domain was smaller than the area AXa for the active domain (mean ratio AXa/AXi = 1.9 ± 0.8 SD, range 1.0-4.3). The signed rank test showed a highly significant (P r(Xi) demonstrating a generally more elongated structure of Xa. For 3D analysis a confocal scanning laser fluorescence microscope (CSLFM) was used. Ten to 20 light optical sections (PI-image, FITC-image) were registered with equal spacings (approx. 0.4 m). A thresholding procedure was applied to determine the PI-labeled nuclear and FITC-labeled X-domain areas in each section. Estimated slice volumes were used to compute total nuclear and X-domain volumes. In a series of 35 female nuclei most domains extended from the top to the bottom nuclear sections. The larger of the two X-chromosome domains comprised (3.7 ± 1.7 S.D.)% of the nuclear volume. A mean ratio of 1.2 ± 0.2 SD (range 1.1-2.3) was found for the volumes of the larger and the smaller X-domains in these female nuclei. In a series of 27 male amniotic fluid cell nuclei the relative X-chromosome domain volume comprised (4.0 ± 2.6 S.D.)%. These findings indicate that differences in the 3D expansion of active and inactive X-chromosome domains are less pronounced than previously thought. A current model suggests that chromosome domains consist of a compact core surrounded by loosely coiled outer chromatin fiber loops. The latter fraction may be considerably larger in Xa- as compared to Xi-domains. We suggest that the interactive outlining procedure used in the 2D analyses included the loosely structured domain periphery more accurately, while the threshold algorithm applied to light optical sections delineated the more compact core of the domains, leading to smaller and more similar volume estimates of Xa and Xi. Present limitations of nuclear and chromosome domain volume measurements using confocal laser scanning microscopy are discussed

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

Cells of synchonized and of asynchronously growing cultures of a V79 Chinese hamster line were microirradiated with a low poweer 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 microirradation, 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 lesions rather than on their distribution in the chromatin. The results are consistent with the prediction of a “factor depletion model” wich assumes that in a given cell, GCS takes place both in irradiated and non-irradiated chromosomes of the total number of daughter strand-repair sites supasses a threshold value

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

Multicolour interphase cytogenetics: 24 chromosome probes, 6 colours, 4 layers

From the late 1980s onwards, the use of DNA probes to visualise sequences on individual chromosomes (fluorescent in-situ hybridisation - FISH) revolutionised the study of cytogenetics. Following single colour experiments, more fluorochromes were added, culminating in a 24 colour assay that could distinguish all human chromosomes. Interphase cytogenetics (the detection of chromosome copy number in interphase nuclei) soon followed, however 24 colour experiments are hampered for this application as mixing fluorochromes to produce secondary colours produces images that are not easily distinguishable from overlapping signals. This study reports the development and use of a novel protocol, new fast hybridising FISH probes, and a bespoke image capture system for the assessment of chromosome copy number in interphase nuclei. The multicolour probe sets can be used individually or in sequential hybridisation layers to assess ploidy of all 24 human chromosomes in the same nucleus. Applications of this technique are in the investigation of chromosome copy number and the assessment of nuclear organisation for a range of different cell types including human sperm, cancer cells and preimplantation embryos