Physical methods sensitive to DNA and protein conformation and structure will be used to characterize the structure and conformation of chromatin from the naturally mitotically synchronous slime mold Physarum polycephalum. Chromatin from this organism has been shown to possess virtually all the features of that from higher eukaryotes. Specific investigations will include the following. I. Principal physical methods will include flow birefringence, circular dichroism and high resolution theraml denaturation. This laboratory is especially well qualified on the basis of experience and facilities to utilize these methods in the studies planned. (A) Chromatin will be characterized as an explicit function of cell cycle. These studies will provide information on how chromatin structure changes with specific phases of biological activity, and hence will provide a higher biological """"""""resolution"""""""" than past studies on transcriptionally inactive bulk chromatin. (B) A novel method will be used to fractionate and concentrate actively transcribing regions in the amounts required for the structural studies planned. This method is based upon the correlation between core histone hyperacetylation and transcriptional activity, and involves replacement of dynamic modification groups by the acetate analog, thioacetate, followed by fractionation using affinity chromatography. These studies may provide the first direct structural and conformational characterization of transcriptionally active or ready-to-transcribe """"""""activated"""""""" chromatin. II. Additional studies will characterize further the role of histone hyperacetylation as a requirement for chromatin unfolding and gene control, and the role of specific DNA base sequences in the possible preferential placement of nucleosomes. (A) Preliminary studies have suggested that histone hyperacetylation may moderate the localized nature of chromatin unfolding. Additional studies on Physarum chromatin are planned to define further this concept. (B) Preliminary studies have suggested that the CAC/GTG triplet may play a role in nucleosome phasing. Additional studies are planned to characterize further this phenomenon.
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