Chromatin structure.? ? We are interested in the biophysical properties and structure of native chromatin fragments. The chicken folate receptor and beta-globin gene loci are ideal for such structural studies in that (i) the region possesses both condensed and transcriptionally active chromatin and (ii) the system has been extensively studied in terms of gene regulation, allowing us to relate the overall chromatin structure to transcription. The condensed chromatin region, spanning 16 kbp of DNA flanked by the developmentally regulated folate receptor and beta-globin genes, can be released from the cell nucleus with the restriction enzyme HpaII. We have previously analyzed the hydrodynamic properties of this condensed chromatin fragment and showed that it is an extended rod. This provides insights into the structure of heterochromatin, found interspersed within various genomes.? ? The biophysical and biological tools developed in these studies have allowed us to further expand our investigation. Using an erythroid precursor cell line (6C2), we have analyzed a 16 kbp region of the transcriptionally poised beta-globin gene locus and showed that this chromatin fragment also appears to be an extended rod. In order to circumvent some of the problems associated with single nucleotide polymorphisms observed in chicken erythrocytes, we have further dissected the beta-globin gene cluster into a series of five distinct chromatin fragments ranging from 2.1 to 8.0 kbp in size. We are currently comparing the fragments released from 6C2 cells with those released from 10-day old and adult chicken erythrocytes. As the beta-globin genes are transcribed in 10-day old erythrocytes but inactive in adult erythrocytes, these studies will allow us to relate the structures of heterochromatin with those of transcriptionally active and inactive chromatin.? ? ? Macromolecular assemblies.? ? In collaboration with members of the Laboratory of Molecular Biology, and other laboratories, protein and protein-nucleic acid assemblies have been characterized in terms of their shape, stoichiometry and affinity of interaction using hydrodynamic methods. These studies extend the biochemical and structural investigations and provide important mechanistic information. A case in point is provided by the recently published work on a 17-residue beta-hairpin carried out in collaboration with Dr. William Eaton and other members of the Laboratory of Chemical Physics. The laser temperature jump kinetic data were consistent with the sedimentation equilibrium thermodynamics describing the reversible monomer-trimer self-association. Together these studies provide information on the folding of the beta-hairpin, namely the support for a zipper mechanism in which structure formation is initiated at the turn and propagated by the sequential formation of cross-strand hydrogen bonds and hydrophobic cluster. This result provides important insights into the process of protein folding.
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