This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. In order to study the role of chromatin proteins in gene expression and silencing, we are analyzing chromatin proteins at the ribosomal DNA (rDNA) locus in budding yeast. Ribosomes are required for the fundamental process of protein synthesis, and their expression is regulated according to how much proteins cells need. This in turn depends on how aggressively they are dividing. In wild type yeast growing exponentially, only about half the ~150 tandemly repeated rDNA genes are active at any time, but as growth plateaus all of the repeats become silenced. Since the repeats have the same sequence, differences in behavior between them must be caused by differences in associated factors. This project has three specific aims. First we will purify native rDNA chromatin from yeast cells. Second we will identify the bound proteins, map their locations along the rDNA, and determine how they are modified using mass spectrometry. Third we will determine differences in chromatin composition and modifications between rDNA that is ?on? (transcribed) and rDNA that is off (silent), and describe how their distributions change as the locus is progressively activated or silenced. The ultimate goal is not only a map of active and silenced rDNA chromatin (including histone modifications), but also a dynamic model of rDNA activation and silencing.
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