The theme of this project is the application fluorescence in situ hybridization (called fluorescence hybridization) to the cytogenetic analysis of normal and malignant cells. Selected chromosomes in metaphase spreads and interphase nuclei will be fluorescently stained with nucleic acid probes. These probes will be of two types: chromosome-specific repetitive sequences, which bind to extended subregions of the target chromosome, usually near the centromeres; and collections of unique sequences (called composite probes) selected so that their binding is distributed along the entire length of a chromosome. We have available repetitive probes for chromosomes 1, 7, 9, 11, 18, X, and Y; and as part of this project will produce composite probes 7, 9, and 22. Multiple probes will be used simultaneously, permitting staining of several chromosomes in the same metaphase spread or interphase nucleus with distinct colors. Specifically we will: 1) Investigate the degree to which the relative positions of chromosomes in interphase nuclei are deterministically established, and how this order might vary with cell cycle position and cell type. Three dimensional reconstructions of chromosome positions in nuclei will be produced by optical sectioning. 2) Use chromosome-specific probes to investigate the occurrence of aneuploidy of chromosomes 7 and Y in clear cell carcinoma of the kidney, emphasizing in particular studies of karyotypic heterogeniety in cultured tumor cells and tissue sections. 3) Use probes for the sex chromosomes to detect early recurrence of leukemia in patients that have received bone marrow transplants from donors of the opposite sex. 4) Use composite probes for chromosomes 9 and 22 to detect translocations between these chromosomes in cases of chronic myelogeneous leukemia. The two chromosomes will be stained different colors, and the signature of the translocation in metaphase spreads will be the presence of bicolored chromosomes. We will investigate the possibility of detecting this translocation in interphase nuclei. The approach to cytogenetic analysis has tremendous clinical potential because of the distinctiveness of chromosome staining, which simplifies identification of abnormalities in metaphase spreads; and the possibility for analysis in interphase nuclei in some cases, which may eliminate the need for time consuming cell culture. We anticipate that the results of our research will lead to clinical applications in oncology in the near future.
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