The basic objectives of this proposal are: (1) to determine details of the internal structure of nucleosomes, structural subunits of chromatin, such as the major order of histones along DNA and the location of specific histone-DNA and histone-histone contact sites; (2) to investigate how in vivo histone modifications, histone subspecies variations, and certain non-histone proteins alter the basic structure of nucleosomes or chromatin in relation to active gene structure. Electrophoresis techniques have been developed for resolving and isolating nucleoprotein fragments of chromatin resulting from nuclease digestion, including subnucleosomal particles containing subsets of histones that arise from cleavages at specific sites within nucleosomes. These techniques will be used to aid in characterizing products obtained after modification of DNA phosphate groups in pure nucleosomes with ethylnitrosourea to map histone contact points and after treatment of nucleosomes with crosslinking reagents. The conformational properties and reassociation of subnucleosomes will be investigated as models for nucleosome structural features. Nucleosomes and other small fragments released at different stages of digestion of mouse myeloma nuclei with DNase I and staphylococcal nuclease will be fractionated and analyzed to determine non-histone proteins, histone modifications and variations, DNA sequence complexity, and content of active genes.