Localization of T-antigen on simian virus 40 minichromosomes by immunoelectron microscopy.
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.
D000818:Animals; D000911:Antibodies, Monoclonal; D000936:Antigen-Antibody Complex / Q000032:analysis; D000952:Antigens, Polyomavirus Transforming; D000957:Antigens, Viral, Tumor / Q000235:genetics*; D002460:Cell Line; D002522:Chlorocebus aethiops; D002843:Chromatin / Q000648:ultrastructure*; D002875:Chromosomes / Q000648:ultrastructure; D007106:Immune Sera; D007668:Kidney; D008854:Microscopy, Electron; D011494:Protein Kinases / Q000235:genetics*; D013539:Simian virus 40 / Q000201:enzymology / Q000235:genetics* / Q000648:ultrastructure; D014764:Viral Proteins / Q000235:genetics*
D000911:Antibodies, Monoclonal; D000936:Antigen-Antibody Complex; D000952:Antigens, Polyomavirus Transforming; D000957:Antigens, Viral, Tumor; D002843:Chromatin; D007106:Immune Sera; D014764:Viral Proteins; D011494:Protein Kinases