The research proposed is directed toward investigating several aspects of chromosome structure in relationship to chromosome dynamics both in normal cells and in cells which have been selected because they are resistant to an antitumor agent. We will apply and extend the EM in situ hybridization techniques we have developed in two directions. In order to facilitate identification of specific active genes in Miller spreads we are extending electron microscopic in situ hybridization techniques to detect hybrids between probes labeled with biotin-substituted nucleotides and nascent transcripts detected via reaction with a ligand adsorbed to colloidal gold particles. Ultrastructural studies will employ this technique to investigate three systems. The structure of chromatin surrounding heat shock loci 87A, 87C and 67B in Drosophila melanogaster will be analyzed in order to relate it to nuclease digestion studies of the same regions; amplifying chorion genes in ovarian follicle cells of Drosophila will be located and studied ot analyze features of an intrachromosomal amplification system; and the structure and activity of double minute chromosomes containing dihydrofolate reductase genes will be investigated. In the latter system transcript hybridization will be applied to locate genes during the generation of double minutes. These experiments will provide information on features of selective replication which results in antitumor resistance. A second set of studies focuses on a characterization of features of inactive X chromosome (Barr bodies) in human fibroblasts. Chromosomal proteins will be fractionated and compared between cells possessing none, one or several Barr bodies. In addition, penta X chromosomal proteins will be used to generate a monoclonal library in order to search for minor inactive X-specific polypeptides. Using cloned X-linked probes Barr body enrichment will be attempted by selective cleavage of enchromatin and subsequent fractionation of resistant material for further analysis. These studies, in concert with those in progress by ourselves and others should provide information on molecules associated with the maintenance of the inactive state and should provide reagents necessary to study the sequence of events which occur during inactivation, a model system for differentiation.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
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Molecular Biology Study Section (MBY)
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University of California Irvine
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Narayanswami, S; Doering, J L; Fokta, F J et al. (1995) Chromosomal locations of major tRNA gene clusters of Xenopus laevis. Chromosoma 104:68-74
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Dvorkin, N; Clark, M W; Hamkalo, B A (1991) Ultrastructural localization of nucleic acid sequences in Saccharomyces cerevisiae nucleoli. Chromosoma 100:519-23

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