Summary.A method of in situ hybridization for visualizing individual human chromosomes from pter to qter, both in metaphase spreads and interphase nuclei, is reported. DNA inserts from a single chromosomal library are labeled with biotin and partially preannealed with a titrated amount of total human genomic DNA prior to hybridization with cellular or chromosomal preparations. The cross-hybridization of repetitive sequences to nontargeted chromosomes can be markedly suppressed under appropriate preannealing conditions. The remaining single-stranded DNA is hybridized to specimens of interest and detected with fluorescent or enzymelabeled avidin conjugates following post-hybridization washes. DNA inserts from recombinant libraries for chromosomes 1, 4, 7, 8, 13, 14, 18, 20, 21, 22, and X were assessed for their ability to decorate specifically their cognate chromosome; most libraries proved to be highly specific. Quantitative densitometric analyses indicated that the ratio of specific to nonspecific hybridization signal under optimal preannealing conditions was at least 8 : 1. Interphase nuclei showed a cohesive territorial organization of chromosomal domains, and laserscanning confocal fluorescence microscopy was used to aid the 3-D visualization of these domains. This method should be useful for both karyotypic studies and for the analysis of chromosome topography in interphase cells.

Summary. 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 readily 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 fllrther 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.

Specific chromosome domains in interphase nuclei of neurons and glia were studied by three-dimensional (3-D) reconstruction of serial optical sections from in situ hybridized human CNS tissue. Overall patterns of centromere organization, delineated with alphoid repeats, were comparable to those seen in mouse, and are clearly conserved in mammalian evolution. Cloned probes from other individual chromosome domains were used to define interphase organization more precisely. Homologous chromosomes were spatially separated in nuclei. In large neurons, probes specific for 9q12, or 1q12 showed that at least one homolog was always compartmentalized together with centromeres on the nucleolus, while the second signal either abutted the nucleolus or was on the nuclear membrane. A telomeric Yq12 sequence also localized together with perinucleolar centromeres in a completely non-Rabl orientation. In astrocytes, these three chromosome regions were on the membrane and not necessarily associated with nucleoli. Therefore there are different patterns of interphase chromosome organization in functionally distinct cell types. In contrast to the above domains, a 1p36.3 telomeric sequence embedded in a large Alu-rich and early replicating chromosome region, was always found in an interior euchromatic nuclear compartment in both neurons and glial cells. In double hybridizations with 1q12 and 1p36.3 probes, 1p arms were clearly separated in all cells, and arms projected radially into the interior nucleoplasm with non-Rabl orientations. There was no absolute or rigid position for each 1p arm with respect to each other or to the major dendrite, indicating that individual chromosome arms may be dynamically positioned even in highly differentiated cell types. We suggest that centromeric and other highly repeated non-transcribed sequence domains may act as general organizing centers for cell type specific interphase patterns that are conserved in mammalian evolution. Such centers would allow selected groups of chromosome arms to extend into (and contract from) an interior, presumably transcriptionally active, nuclear compartment.

The position of selected chromosomes was assessed in samples of normal and epileptic human cortex with biotinylated probes specific for individual chromosome domains. Optical sectioning provided a rapid method for three-dimensional resolution of in situ hybridization signals in interphase cells, and solid models were reconstructed from digitized images for detailed rotational studies. There was a dramatic repositioning of the X chromosome in neurons of both males and females in electrophysiologically defined seizure foci. Other chromosomes (1, 9, and Y) showed more subtle positional changes. Specifically altered nuclear patterns involving the X chromosome may become established and create the genetic memory for intractable seizure activity.