The long-range goal of this project is to understand parameters which determine spatial and functional organization of DNA in the nucleus. Specifically, the work will identify chromosomal DNA sites which are anchored to immobile objects in yeast Saccharomyces cerevisiae. DNA immobilization by specific DNA sequences may represent a fundamental unit of chromosome architecture; DNA anchors may segregate chromosomal DNA into independent structural domains for DNA packaging, regulated gene expression and positioning within the nucleus. DNA anchoring sequences and associated proteins will be characterized using a newly developed molecular biological technique. The approach employs site specific recombination in vivo to generate well-defined DNA rings which contain a single promoter and a DNA fragment to be tested. Transcription of anchored DNA rings ina yeast topoisomerase mutants leads to diagnostic changes in DNA topology, in accordance with the twin domain model of transcriptional supercoiling. models of DNA organization based on analyses of biochemically manipulated chromosomes, termed nuclear scaffold, argue that DNA is arranged into loops with specific DNA segments anchoring the bases of the loops to an organizational framework. The excision methodology will be used to investigate whether sequences which bind yeast scaffold preparations function as DNA anchors in vivo. Scaffold attachment regions (SARs) to be examined include chromosomal components such as telomeres, silencers, centromeres and origins of replication. DNA anchor formation. The cell- cycle dependent anchoring of centromeres and replication origins will be determined in synchronized cell cultures. A selection system will be developed to isolate sequences with DNA anchoring activity from libraries of the sequenced yeast Chromosome III. The DNA loop structure of specific chromosomal regions will be determined by mapping the DNA anchoring elements which reside within the regions. The influence of genomic DNA anchors on plasmid partitioning and gene expression will be examined.
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