Chromosome mapping by FISH to metaphase and interphase nuclei. Final report

Abstract

The overall specific aims of this project were: 1) to determine the large-scale structure of interphase and metaphase chromosomes, in order to establish new capabilities for genome mapping by fluorescence in situ hybridization (FISH). to detect chromosome abnormalities associated with genetic disease and map DNA sequences relative to them in order to facilitate the identification of new genes with disease-causing mutations .to establish medium resolution physical maps of selected chromosomal regions using a combined metaphase and interphase mapping strategy and to corroborate physical and genetic maps and integrate these maps with the cytogenetic map. to analyze the polymorphism and sequence evolution of subtelomeric regions of human chromosomes. to establish a state-of-the art HSH and image processing facility in the department of Molecular Biotechnology, University of Washington, in order to map RNA sequences rapidly and accurately to benefit the Human Genome Project. 2) 4) )We achieved each of these aims during the course of this project. Details of our results are given in the following sections, which are numbered to correspond with the specific aims. Organization of chromatinThis project has contributed to new insight into the organization of human GO interphase and metaphase cells. The results defined an appropriate role for FISH in genome analysis. It was well known that DNA is tightly packaged in metaphase cells. We established that DNA sequences can only be reliably ordered along the centromere-telomere axis if they are separated by > 0.5 Mbp, while an even higher threshold may apply at the ends of chromosomes . Using a combination of interphase (untreated and borate-treated), a correlation between physical distance and genomic separation can be obtained at distances > 50 kbp.chromosome structure in part as a consequence of this DOE funding. Because chromatin is more loosely packed in interphase, resolution of DNA sequences is enhanced to -50 kbp. We made detailed measurements of the distance in interphase between probes separated by 0.05 to 200 Mbp on the same chromosome by taking advantage of mapped probe resources flowing from the human genome project. Our measurements led to a new model of chromosome organization (Sachs et al., 1995;Yokota et al., 1995b). We found that the relationship between mean-square interphase distance and genomic separation has two linear phases with a transition at -2 Mbp. On the first level, DNA appears to be randomly folded as a highly flexible polymer, but is constrained in loops several Mbp in size. This level of organization accounts for the first steep phase of the relationship, a phenomenon that can be exploited to produce detailed genomic maps. We demon-The Trask laboratory has been at the forefront of the study of the structure of interphase