Colorado PROFILES, The Colorado Clinical and Translational Sciences Institute (CCTSI)
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The objective of the proposed research is to characterize the chromatin structure of a eukaryotic ribosomal RNA gene (rDNA). The extrachromosomal rDNA in the macronucleus of the ciliated protozoan, Tetrahymena, will be used as a model system. The investigation will focus on four regions of the rDNA: the transcriptional initiation and termination regions, the origins of replication, and the telomeres (the ends of the minichromosomes). Various nucleases (micrococcal nuclease, DNase I, DNase II, S1 nuclease) and MPE-Fe(II), a chemical DNA-cleavage agent, will be used as probes for nucleosomes and for other DNA-protein interactions. Comparison of active and inactive sets of genes will allow the establishment of structure-function relationships (e.g., a change in chromatin structure near the promoter that accompanies transcriptional activation). A technique will be developed to extend the resolution of the protein mapping to the nucleotide sequence level. Ribosomal gene chromatin (rChromatin) will be purified, and its DNA-protein interactions will be probed to determine whether they have been preserved through the isolation procedure. Attempts will be made to isolate proteins that bind in sequence-specific manner to the rDNA. In combination with in vitro transcription experiments, the structural studies should allow us to determine the roles of various nonhistone (and perhaps histone) proteins in rRNA gene expression.
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