The research proposed is aimed at understandng the chemical and cytological features of human chromosome structure, replication and the response to DNA damage. Fluorescent dyes, including new dyes with red fluorescence, will be used singly and in pairs, the latter sensing energy transfer, together with flow cytometry, to characterize better the DNA content, DNA replication kinetics, and chromatin condensation of normal and genetically abnormal cells (from patients with diseases (Fanconi's anemia (FA) and ataxia telangiectasia (AT), associated with a predisposition for the development of cancer), before and after DNA damage. Cytochemical detection of BrdU incorporation will contribute to this work and to experiments focusing on sister chromatid exchange (SCE) formation. Cytological and biochemical studies of factors, e.g. DNA methylation, which influence SCE formation, a sensitive index of cellular exposure to environmental mutagens, will be performed, and controlled damage(e.g. with psoralens) of DNA used to test hypotheses for the mechanism of SCE formation and to search for biochemical evidence of DNA interchange associated with SCE formation in defined DNA sequences. Cloned DNA probes complementary to highly amplified DNA sequences, localizable at a cytological level, and employed in Southern blots will be important for this work. Biochemical studies probing the repair defects in FA and AT are planned, as are DNA transformation experiments, using human DNA and rodent cells with DNA repair defects modelling these diseases, with the goal of better diagnosis and understanding of the genetic changes and molecular pathology of FA and AT. Analysis of DNA methylation, chromatin condensation, and DNA replication kinetics will also be extended to a molecular level to study the control of gene expression associated with human X chromosome inactivation. Several cloned genomic and cDNA probes, with homology to the human X, will be employed, together with quantitative Southern blotting, selective photodegradation of BrdU-substituted DNA sequences, northern blotting, digestion of DNA with isoschizomer restriction enzyme pairs, DNA'se digestion of chromatin, and protein analysis on flow sorted active and inactive human X chromosomes to investigate the molecular correlates and mechanism of X inactivation, particularly as they vary between different regions of the X or different cell types. The results should aid diagnosis and understanding of the phenotypic consequences of human X chromosome abnormalities.
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