Chromatin structure and architecture. DNA within the cell nucleus is packaged into chromatin and a variety of models currently describe the structure of the condensed 30 nm chromatin fiber observed in vitro. However, evidence for this structure in vivo is lacking, except in specialized cells such as mature avian erythrocytes in which all of the chromatin is essentially inactive. We are interested in understanding the organization of DNA within condensed chromatin in vivo, as well as the topological constraints imposed on its higher order by organizing proteins such as CTCF and cohesin. We are developing high resolution chromosome capture conformation assays utilizing native chromatin fragments, such as the previously studied condensed heterochromatin flanked by the developmentally regulated folate receptor and beta-globin genes. These studies will allow us to better understand the structure of the chromatin fiber in vivo, thus providing insight in the relations between chromatin structure and essential processes such as gene expression and DNA replication. Macromolecular assemblies of biological interest. Biological assemblies have been characterized in terms of their shape, stoichiometry and affinity of interaction using hydrodynamic methods. These studies complement current investigations, as evidenced by recent work by Schmidt et al. (2016). In this work, a simple method using electron paramagnetic resonance (EPR) is developed to determine the populations of protein oligomers in solution. Such studies on the p66 subunit of HIV-1 reverse transcriptase demonstrate that the presence of glycerol results in a shift of the monomer-dimer equilibrium. We utilized analytical ultracentrifugation to characterize the p66 monomer-dimer equilibrium and confirm the surprising EPR observations, demonstrating the effects of glycerol. Analytical ultracentrifugation is one of the primary tools used for the hydrodynamic studies described. In collaboration with colleagues from the NIH, and others, we have published a textbook describing current methodology in the field (Schuck et al., 2016).
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