This proposal has four long-range objectives: 1) to determine systematically the topological distribution of individual chromosomal domains within the interphase nuclei of human cells; 2) to establish unambiguously whether these chromosomal domains exhibit a definable three-dimensional structure with precise spatial interrelationships; 3) to determine to what extent does the chromosomal architecture of the nucleus change during the mitotic cell cycle, normal cellular differentiation and neoplastic transformation and 4), to develop a rapid method of interphase cytogenetics suitable for the diagnosis of specific genetic and malignant diseases. Specific research goals are: 1) to clone and characterize novel sets of DNA containing sequences that hybridize uniquely to a single type of human chromosome. These sequence subsets will be prepared from parent libraries of individual chromosomes, sorted by flow-cytometry, using subtractive hybridization with appropriate biotinylated driver probes and affinity fractionation over avidin- metal chelate resins. The specificity of each affinity-enriched DNA pool will be tested by dot-hybridization to multiple chromosomal DNA libraries, by in situ hybridization to metaphase chromosome spreads and by Southern blot analysis of somatic cell hybrid panels, 2) to identify individual chromosome domains within selected human cell populations and tissues by in situ hybridization with one or more sets of chromosome-unique DNA, each set being labeled with a different reporter molecule which can be detected independently with reporter-specific antibodies tagged with distinct, high quantum yield fluorochromes, 3) to establish the potential utility of chromosome-specific DNA probe sets for the detection of numerical and/or structural chromosome aberrations in interphase cells 4) to assemble a computer-based fluorescence imaging system and associated software programs suitable for the reconstruction of three-dimensional structures from multiple optical sections and 5) to apply these analytical tools to elucidate the nuclear architecture of chromosomes in human cells at various stages of the cell cycle, in cells of different developmental lineages, in natural tumors and cells transformed by viral or chemical agents.
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