Selective DNA binding of the human cellular myb protein isolated by immunoaffinity chromatography using a monoclonal antibody.
The bacterially expressed v-myb protein served as antigen for the isolation of several monoclonal antibodies, one of which recognized the human cellular myb protein (p75hu-c-myb) indicating a conserved epitope. The epitope was mapped to amino acid positions 208-232 by the use of several bacterially expressed v-myb proteins with various deletions. Furthermore, a synthetic oligopeptide which had been selected on the basis of its hydrophilicity by computer analysis of the v-myb oncogene (amino acids 213-231) blocked the action of this monoclonal antibody, indicating the immunological significance of this region. The monoclonal antibody allowed efficient purification of the p75hu-c-myb protein by immunoaffinity chromatography. The purified protein binds to double-stranded DNA in vitro in a filter-binding assay. Since the monoclonal antibody does not interfere with DNA binding it allowed analysis of DNA-protein interaction in a modified McKay assay using the purified p75hu-c-myb protein. Specific binding was observed predominantly to one of 12 lambda DNA fragments in vitro in the presence of high molar excess of competing co-polymer poly [d(I:C)]. Enhancer/promoter-like sequences of SV40 were not preferentially recognized.
Bading
H
H
Max-Planck-Institut für Molekulare Genetik, Berlin, FRG.
Hansen
J
J
Moelling
K
K
D000911:Antibodies, Monoclonal / Q000276:immunology; D002846:Chromatography, Affinity; D004268:DNA-Binding Proteins / Q000276:immunology / Q000302:isolation & purification / Q000378:metabolism*; D000939:Epitopes; D006801:Humans; D008970:Molecular Weight; D009856:Oncogene Proteins, Viral / Q000276:immunology; D011518:Proto-Oncogene Proteins / Q000276:immunology / Q000302:isolation & purification / Q000378:metabolism*; D012045:Regulatory Sequences, Nucleic Acid; D013329:Structure-Activity Relationship
D000911:Antibodies, Monoclonal; D004268:DNA-Binding Proteins; D000939:Epitopes; D009856:Oncogene Proteins, Viral; D011518:Proto-Oncogene Proteins