The heirarchy of packing interactions in chromatin begins with nucleosomes and culminates in mitotic chromosome bands. Lower levels of packing are apparently determined by DNA-histone and histone-histone interactions, but higher levels (such as organization of chromatin into 100 kb topological domains) are probably regulated by non-histone proteins. Our long term goal is to understand the role of non-histone proteins in determining mitotic chromosome architecture. The best candidates for structural non-histone proteins are components of the poorly characterized chromosome scaffold fraction (proteins which remain associated after extraction of 99% of the chromosomal DNA and 95% of the protein). We have recently shown that DNA topoisomerase II is a component of this fraction. We will continue to characterize this and other scaffold components. 1) Antibodies (polyclonal, monoclonal, autoimmune) recognizing specific scaffold antigens will be obtained. These will be used to determine the efficiency of recovery of specific antigens in the scaffold fraction, to localize these antigens in intact chromosomes and whole cells by immunofluorescence and immunoelectron microscopy, to quantitate the antigens across the cell cycle and in non-dividing cells by photometry and immunoprecipitation, and to map the interactions between scaffold components by gradient sedimentation and immunoprecipitation. 2) The role of scaffold proteins in chromosome condensation will be assayed by studying the effect of the antibodies on premature chromosome condensation (induced when mitotic and interphase cells are fused). 3) Highly extended prematurely condensed chromosomes will also be used for structural studies. The folding of the chromatin fiber will be examined using fluorescence microscopy coupled with computer image processing methods. 4) The activity of topoisomerase II in mitotic cells will be examined. We describe an in situ assay for DNA gyrase activity in deproteinized mitotic chromosomes. 5) Finally, we describe a developmental system (chicken erythropoiesis) where scaffold proteins are progessively lost. We will examine the changes in chromatin domain structure which accompany this loss in order to deduce the function of various scaffold components in establishment of nuclear structure. Techniques used will include immunofluorescence, immunoblotting, scanning calorimetry and quantitative fluorimetry (used to measure the size of chromatin domains).
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