Localization of T-antigen on simian virus 40 minichromosomes by immunoelectron microscopy

Abstract

SV40 chromatin extracted from 42 h post-infected cells by a modification of the standard Triton X-100-EDTA procedure and purified on neutral sucrose gradients was partially immunoprecipitable by a specific SV40 T-antigen (T-Ag) antiserum. Electron microscopic observations of spread minichromosomes were made after labelling by the indirect colloidal gold immunological method using monoclonal antibodies specific for the SV40 T-Ag. In 1-2% of morphologically mature minichromosomes the labelling corresponding to tightly bound T-Ag was localized within the nucleosome-free region near one of its borders. Mapping with three single-cut restriction endonucleases: BamHI, EcoRI and BglI localized the labelling near to, or at the origin of, replication. In addition, we observed that the T-Ag specific antibodies were linked to a DNA-bound particle when the region was not masked by a large clump of antibodies. The variable size of this particle led us to suggest that it might be a complex of T-Ag with other proteins.