Detection of complete and partial chromosome gains and losses by comparative genomic in situ hybridization

Comparative genomic in situ hybridization (CGH) provides a new possibility for searching genomes for imbalanced genetic material. Labeled genomic test DNA, prepared from clinical or tumor specimens, is mixed with differently labeled control DNA prepared from cells with normal chromosome complements. The mixed probe is used for chromosomal in situ suppression (CISS) hybridization to normal metaphase spreads (CGH-metaphase spreads). Hybridized test and control DNA sequences are detected via different fluorochromes, e.g., fluorescein isothiocyanate (FITC) and tetraethylrhodamine isothiocyanate (TRITC). The ratios of FITC/TRITC fluorescence intensities for each chromosome or chromosome segment should then reflect its relative copy number in the test genome compared with the control genome, e.g., 0.5 for monosomies, 1 for disomies, 1.5 for trisomies, etc. Initially, model experiments were designed to test the accuracy of fluorescence ratio measurements on single chromosomes. DNAs from up to five human chromosome-specific plasmid libraries were labeled with biotin and digoxigenin in different hapten proportions. Probe mixtures were used for CISS hybridization to normal human metaphase spreads and detected with FITC and TRITC. An epifluorescence microscope equipped with a cooled charge coupled device (CCD) camera was used for image acquisition. Procedures for fluorescence ratio measurements were developed on the basis of commercial image analysis software. For hapten ratios 4/1, 1/1 and 1/4, fluorescence ratio values measured for individual chromosomes could be used as a single reliable parameter for chromosome identification. Our findings indicate (1) a tight correlation of fluorescence ratio values with hapten ratios, and (2) the potential of fluorescence ratio measurements for multiple color chromosome painting. Subsequently, genomic test DNAs, prepared from a patient with Down syndrome, from blood of a patient with Tcell prolymphocytic leukemia, and from cultured cells of a renal papillary carcinoma cell line, were applied in CGH experiments. As expected, significant differences in the fluorescence ratios could be measured for chromosome types present in different copy numbers in these test genomes, including a trisomy of chromosome 21, the smallest autosome of the human complement. In addition, chromosome material involved in partial gains and losses of the different tumors could be mapped to their normal chromosome counterparts in CGH-metaphase spreads. An alternative and simpler evaluation procedure based on visual inspection of CCD images of CGH-metaphase spreads also yielded consistent results from several independent observers. Pitfalls, methodological improvements, and potential applications of CGH analyses are discussed

Higher concentrations of histone macroH2A in the Barr body are correlated with higher nucleosome density

AbstractHistone macroH2A, which is a subtype of histone H2A, possesses a histone H2A-like portion fused to a relatively long non-histone portion. MacroH2A has been shown to associate preferentially with the inactive X chromosome [1]. To investigate the specificity of this association, the nuclear distribution of macroH2A was compared with that of regular core histones. In normal human female fibroblasts, all anti-histone antibodies that were tested (including anti-macroH2A antibody) preferentially labeled the inactive X chromosome. Moreover, when expressed as green fluorescent protein (GFP) fusions, both histone H2A and macroH2A were concentrated in the Barr body. These data clearly show the presence of a higher density of nucleosomes in the inactive X chromosome. Accordingly, the specificity of the macroH2A association with the inactive X chromosome should be reconsidered. While investigating the role of macroH2A, we found that the proximity of the non-histone region of macroH2A to a promoter could lead to a specific repression of transcription, suggesting that the incorporation of macroH2A into chromatin might help to establish the stable pattern of gene expression in differentiated cells

3D-Voronoi Diagramme zur quantitativen Bildanalyse in der Interphase-Cytogenetik

Um die Anordnung von Chromosomen in Zellkernen der Interphase zu untersuchen, wurde ein Verfahren aus der Computergeometrie adaptiert. Dieser Ansatz basiert auf der Zerlegung von dreidimensionalen Bildvolumen mithilfe des Voronoi-Diagramms in konvexe Polyeder. Die graphenorientierte, geometrische Struktur dieses Verfahrens ermöglicht sowohl eine schnelle Extraktion von Objekten im Bildraum als auch die Berechnung morphologischer Parameter wie Volumina, Oberflächen und Rundheitsfaktoren. In diesem Beitrag wird exemplarisch die dreidimensionale Morphologie von XChromosomen in weiblichen Interphasezellkernen mithilfe dieser drei Parameter untersucht. Um diese Zellkerne mit lichtoptischen Methoden zu untersuchen, wurden die Territorien der X-Chromosomen mit einem molekularcytogenetischen Verfahren fluoreszierend dargestellt. Zur Unterscheidung des aktiven und inaktiven X-Chromosoms wurde das Barr-Körperchen zusätzlich markiert und mithilfe eines Epifluoreszenzmikroskops, ausgerüstet mit einer CCD-Kamera, aufgenommen. Anschließend wurden 1 2 - 2 5 äquidistante, lichtoptische Schnitte der X-Chromosomenterritorien mit einem konfokalen Laser Scanning Mikroskop (CLSM) aufgenommen. Diese lichtoptischen Schnitte wurden mithilfe des Voronoi-Verfahrens segmentiert und analysiert. Methoden aus der Computergraphik wurden zur Visualisierung der Ergebnisse eingesetzt. Es konnte gezeigt werden, daß mithilfe des Voronoi-Verfahrens Chromosomen- Territorien anhand der morphologischen Parameter zuverlässig beschrieben werden können

Etude des fonctions de transcription et dépissage au cours du choc thermique

L'exposition des cellules à un stress induit une réponse ubiquitaire, instantanée et transitoire visant à assurer la survie des cellules, qui se traduit par l'activation d'une famille de gènes codant pour des protéines hautement conservées : les HSPs (heat shock proteins). L'expression des gènes hsp au cours du stress est contrôlée par le facteur de transcription HSF1 (heat shock factor 1), qui lorsqu'il est activé par un stress se lie non seulement au promoteur des gènes hsp dont il active la transcription, mais s'accumule également dans des structures nucléaires particulières distinctes des gènes hsp et appelées " granules nucléaires de stress ". Ces structures sont temporaires et se forment au niveau de la région hétérochromatique 9q12 chez l'Homme par une liaison directe du facteur HSF1 à des séquences de type satellite III. Nous montrons ici que les granules de stress sont le siège d'une transcription active par l'ARN polymérase II des séquences répétées satellite III.GRENOBLE1-BU Sciences (384212103) / SudocSudocFranceF

Comparison of chromosomal imbalances in neuroendocrine and non-small-cell lung carcinomas.

International audienceLung carcinomas are represented by non-small-cell lung carcinomas (NSCLC) and neuroendocrine carcinomas (NE) which differ in their clinical presentation and prognosis. We used comparative genomic hybridization (CGH) to characterize and compare the chromosomal pattern of 11 NSCLC and 11 high-grade NE lung carcinomas. Overall, the total number of aberrations was higher in NSCLC than in high-grade NE lung tumors (p < 0.05) and gains predominated over losses in NSCLC (p < 0.0003). Gains common to both lung tumor phenotypes were detected in 1p, 1q, 3q, 5p, 6p, 8q, 12, 17q, 19p, 19q, 20p, 20q, and X, whereas common losses were found in 2q, 3p, 4p, 4q, 5q, 8p, 9p, 10p, 11p, 11q, 13q, and 17p. Major gains on 18q and losses on 2p and 16q were exclusively detected in high-grade NE lung tumors. On the other hand, major gains on 2p and 15q and losses on 21q were found only in NSCLC. Furthermore, gains within 22q11-q12 and 7p12-p15 were associated with NSCLC (p < 0.05). The differences in the pattern and distribution of genetic changes observed in NSCLC as opposed to high-grade NE lung carcinomas suggest the existence of distinct tumorigenic pathways between these two major classes of lung tumors