MONOCLONAL ANTIBODY PROBES FOR CHROMATIN ASSOCIATED PROTEINS
Abstract
Nonhistone chromatin proteins, long thought vital for the processes of eukaryotic biology by most investigators, have remained little more than a definition in most textbooks. The lack of molecular probes for the nonhistones has hampered progress toward understanding the proteins. This thesis is an investigation of the potential for monoclonal antibodies to at least partially fill this void. Antibodies to chicken erythrocyte HMG 1 and 2a defined 5 distinct antigenic determinants on the two proteins. The antibodies were able to disrupt the binding of single stranded DNA to HMG 1 and 2a to varying degrees. At a relatively low concentration of an HMG 2a antibody, the HMG 2a:DNA interaction was eliminated but the HMG 1:DNA interaction was unaffected. The evolutionary conservation of the determinants was also investigated. One HMG determinant was found only in birds and reptiles whereas another occurred in all vertebrates examined (mammals, birds, reptiles, amphibia, fish). Some of the antigenic determinants were differentially accessible to their respective antibodies when assayed in a chromatin context. HMG 2a was shown to be highly enriched in a mononucleosome fraction generated by micrococcal nuclease digestion of reticulocyte nuclei. One antibody was generated, rather unexpectedly, to the erythrocyte specific histone, H5. Procedures were developed for the specific binding of nuclease disrupted chromatin to an immunoaffinity matrix of the H5 antibody. Antisera raised in mice to chromatin preparations from different chicken tissues reacted preferentially with the preparation from the tissue used for immunization. A large fraction of the monoclonal antibodies obtained from mice immunized with an erythrocyte chromatin preparation recognized tissue specific chromatin associated proteins. However, it appears from the results of others in our laboratory that these antibodies may be directed to proteins present on the erythrocyte plasma membrane; the association of the antigens with chromatin may be artefactual.
Degree
Ph.D.
Subject Area
Biology
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