This proposal extends research which exploits the resolution of the electron microscope (EM) to study the relationship between the organization and structure of eukaryotic chromatin and chromosomes and chromosome function. A major emphasis is the continued refinement and development of techniques to map at the ultrastructural level the location of specific DNA sequences with respect to mitotic chromosome organization and to rapidly and definitively identify specific active genes in Miller spread specimens. These goals will be pursued by combining in situ hybridization of DNA or RNA probes tagged with the nonradioactive labels biotin or 2-acetylaminofluorene (AAF) to preparations on EM grids followed by immunogold staining. This technique will be used to investigate the existence of mouse satellite sequences on the Y chromosome; the molecular basis of chromosomal rosettes; transcription of histone genes in somatic cells of the newt, Notophthalmus viridescens; and distribution of nascent transcripts on Drosophila heat shock genes. The sensitivity of sequence detection will be determined using the dihydrofolate reductase (DHFR) gene and cells with either extrachromosomal or integrated amplified copies, the X-linked human factor VIII gene and bovine papilloma virus-transformed cells. Biotinated nucleotides will be incorporated into cells induced to rereplicate their DNA in order to identify and directly study the structure and frequency of this event with respect to normal replication and transcription. In addition, we will attempt to selectively incorporate bio-dNTPs into chromosomal fragile sites and telomeres. Finally, immunogold labelling will be applied to develop efficient mapping procedures for the location of chromosome-associated proteins. The long term goal of the proposed work is to provide information at the ultrastructural level on chromosome organization which, in conjunction with biochemical and molecular data, should generate a better understanding of the role of chromosome structure in chromosome function in normal and abnormal states.
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