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ALPHA-CHYMOTRYPSIN: A PROBE OF NUCLEOSOME FINE STRUCTURAL TOPOGRAPHY

The protease (alpha)-chymotrypsin, specific for uncharged hydrophobic residues, was used to probe the fine structural topography of the nucleosome core. The consequences of proteolytic cleavages at specific histone sites were assessed in terms of effects upon the overall nucleohistone structural integrity. / Electrophoresis of histones from partially proteolyzed cores on 18% polyacrylamide slab gels containing SDS showed that H3 was rapidly digested by chymotrypsin, yielding a single product (CP1) approximately 112 residues long. Extensive digestion yielded a stable limit pattern of four major histone fragments ranging in size from 102-80 residues long. Comparative electrophoresis performed on gels containing 8 M urea, 5% acetic acid, and 6 mM triton X-100 established that other core histones were essentially resistant to digestion until the majority of H3 was degraded. Second dimension electrophoresis showed that CP1 migrated near H3 in the trition-acid-urea system. Also resolved by second dimension electrophoresis were two additional products that migrated near H4 and CP1 (CP2' and CP3'), respectively, in the SDS-dimension. / High pressure liquid chromatography peptide analyses confirmed that the major chymotryptic product, CP1, was derived from H3 and had an intact carboxy terminus. HPLC analyses of dansyl amino acids derived from CP1 made it possible to assign leucine 20 as the preferential chymotryptic cleavage site in H3. This is the only one of 72 possible chymotryptic sites in core histones that clearly is topographically accessible in native cores. Few and perhaps no sites are accessible in the other core histones. / Cleavage at leucine 20 in H3 promoted structural unfolding and induced changes in the core DNA winding angle. Circular dichroism and Raman spectroscopy, thermal denaturation, DNAse I digestion, and nucleo-histone electrophoresis studies were used to characterize these changes and indicated that non-basic histone-DNA interactions were essential for the maintenance of core structure and stability. / Source: Dissertation Abstracts International, Volume: 43-07, Section: B, page: 2091. / Thesis (Ph.D.)--The Florida State University, 1982.

Identiferoai:union.ndltd.org:fsu.edu/oai:fsu.digital.flvc.org:fsu_74903
ContributorsROSENBERG, NANCY LYNN., Florida State University
Source SetsFlorida State University
Detected LanguageEnglish
TypeText
Format146 p.
RightsOn campus use only.
RelationDissertation Abstracts International

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