Fluorescence in situ hybridization of YAC clones after Alu-PCR amplification

Alu-PCR protocols were optimized for the generation of human DNA probes from yeast strains containing yeast artificial chromosomes (YACs) with human inserts between 100 and 800 kb in size. The resulting DNA probes were used in chromosome in situ suppression (CISS) hybridization experiments. Strong fluorescent signals on both chromatids indicated the localization of specific YAC clones, while two clearly distinguishable signals were observed in ≥90% of diploid nuclei Signal intensities were generally comparable to those observed using chromosome-specific alphoid DNA probes. This approach will facilitate the rapid mapping of YAC clones and their use in chromosome analysis at all stages of the cell cycle

Evidence for a nuclear compartment of transcription and splicing located at chromosome domain boundaries

The nuclear topography of splicing snRNPs, mRNA transcripts and chromosome domains in various mammalian cell types are described. The visualization of splicing snRNPs, defined by the Sm antigen, and coiled bodies, revealed distinctly different distribution patterns in these cell types. Heat shock experiments confirmed that the distribution patterns also depend on physiological parameters. Using a combination of fluorescencein situ hybridization and immunodetection protocols, individual chromosome domains were visualized simultaneously with the Sm antigen or the transcript of an integrated human papilloma virus genome. Three-dimensional analysis of fluorescence-stained target regions was performed by confocal laser scanning microscopy. RNA transcripts and components of the splicing machinery were found to be generally excluded from the interior of the territories occupied by the individual chromosomes. Based on these findings we present a model for the functional compartmentalization of the cell nucleus. According to this model the space between chromosome domains, including the surface areas of these domains, defines a three-dimensional network-like compartment, termed the interchromosome domain (ICD) compartment, in which transcription and splicing of mRNA occurs

Comparative chromosome band mapping in primates byin situ suppression hybridization of band specific DNA microlibraries

A DNA-library established from microdissected bands 8q23 to 8q24.1 of normal human chromosomes 8 (Lüdecke et al., 1989) was used as a probe for chromosomal in situ suppression (CISS-) hybridization to metaphase chromosomes of man and primates including Hylobates lar and Macaca fuscata. Comparative band mapping as first applied in this study shows the specific visualization of a single subchromosomal region in all three species and thus demonstrates that synteny of the bulk sequences of a specific human chromosome subregion has been conserved for more than 20 million years

Detection of chromosome aberrations in metaphase and interphase tumor cells by in situ hybridization using chromosome-specific library probes

Chromosome aberrations in two glioma cell lines were analyzed using biotinylated DNA library probes that specifically decorate chromosomes 1, 4, 7, 18 and 22 from pter to qter. Numerical changes, deletions and rearrangements of these chromosomes were radily visualized in metaphase spreads, as well as in early prophase and interphase nuclei. Complete chromosomes, deleted chromosomes and segments of translocated chromosomes were rapidly delineated in very complex karyotypes. Simultaneous hybridizations with additional subregional probes were used to further define aberrant chromosomes. Digital image analysis was used to quantitate the total complement of specific chromosomal DNAs in individual metaphase and interphase cells of each cell line. In spite of the fact that both glioma lines have been passaged in vitro for many years, an under-representation of chromosome 22 and an over-representation of chromosome 7 (specifically 7p) were observed. These observations agree with previous studies on gliomas. In addition, sequences of chromosome 4 were also found to be under-represented, especially in TC 593. These analyses indicate the power of these methods for pinpointing chromosome segments that are altered in specific types of tumors

A strategy for the characterization of minute chromosome rearrangements using multiple color fluorescence in situ hybridization with chromosome-specific DNA libraries and YAC clones

The identification of marker chromosomes in clinical and tumor cytogenetics by chromosome banding analysis can create problems. In this study, we present a strategy to define minute chromosomal rearrangements by multicolor fluorescence in situ hybridization (FISH) with whole chromosome painting probes derived from chromosome-specific DNA libraries and Alu-polymerase chain reaction (PCR) products of various region-specific yeast artificial chromosome (YAC) clones. To demonstrate the usefulness of this strategy for the characterization of chromosome rearrangements unidentifiable by banding techniques, an 8p+ marker chromosome with two extra bands present in the karyotype of a child with multiple anomalies, malformations, and severe mental retardation was investigated. A series of seven-color FISH experiments with sets of fluorochrome-labeled DNA library probes from flow-sorted chromosomes demonstrated that the additional segment on 8p+ was derived from chromosome 6. For a more detailed characterization of the marker chromosome, three-color FISH experiments with library probes specific to chromosomes 6 and 8 were performed in combination with newly established telomeric and subtelomeric YAC clones from 6q25, 6p23, and 8p23. These experiments demonstrated a trisomy 6pter6p22 and a monosomy 8pter8p23 in the patient. The present limitations for a broad application of this strategy and its possible improvements are discusse

Targeting atypical protein kinase C iota reduces viability in glioblastoma stem-like cells via a notch signaling mechanism

In a previous study, Protein Kinase C iota (PRKCI) emerged as an important candidate gene for glioblastoma (GBM) stem-like cell (GSC) survival. Here, we show that PKCι is overexpressed and activated in patient derived GSCs compared with normal neural stem cells and normal brain lysate, and that silencing of PRKCI in GSCs causes apoptosis, along with loss of clonogenicity and reduced proliferation. Notably, PRKCI silencing reduces tumor growth in vivo in a xenograft mouse model. PKCι has been intensively studied as a therapeutic target in non-small cell lung cancer, resulting in the identification of an inhibitor, aurothiomalate (ATM), which disrupts the PKCι/ERK signaling axis. However, we show that, although sensitive to pharmacological inhibition via a pseudosubstrate peptide inhibitor, GSCs are much less sensitive to ATM, suggesting that PKCι acts along a different signaling axis in GSCs. Gene expression profiling of PRKCI-silenced GSCs revealed a novel role of the Notch signaling pathway in PKCι mediated GSC survival. A proximity ligation assay showed that Notch1 and PKCι are in close proximity in GSCs. Targeting PKCι in the context of Notch signaling could be an effective way of attacking the GSC population in GBM