Two major subclasses of simian virus 40 (SV40) large T antigen were separated by zone velocity sedimentation of crude extracts from productively infected cells. These subclasses, which have been shown to differ biologically and biochemically (Fanning et al., 1981), sedimented at 5‐6S and 14‐16S. The amount of T antigen in each form was estimated by complement fixation and by immunoprecipitation of T antigen from extracts of cells chronically labeled with [35S]methionine. Each form of T antigen was tested for specific binding to end‐labeled restriction fragments of SV40 DNA using an immunoprecipitation assay. The 5‐6S and 14‐16S forms of T antigen both bound specifically to DNA sequences in the SV40 HindIII C fragment. The sequences required for binding both forms were localized in the same 35‐bp region of the origin. However, significant differences in binding activity and affinity for specific and nonspecific DNA were demonstrated. These properties suggest that T antigen subclasses may serve different functions in the lytically infected cell.

Two major subclasses of simian virus 40 tumor antigen were prepared from productively infected monkey cells. These subclasses can be distinguished by their sedimentation properties: one tumor antigen form sediments at 5–6 S and the other at 14–16s. The DNA‐binding properties of these subclasses were investigated by two different experimental procedures. In the first procedure, the DNA binding of subclasses of crude tumor antigen, separated by zone velocity sedimentation, were assayed by immunoprecipitation of the DNA‐protein complexes. In the second procedure, the two tumor antigen forms were partially purified by column chromatography and DNA binding was tested in a filter binding assay. Both procedures gave comparable results. (a) The 5–6‐S and the 14–16‐S tumor antigen bound specifically to a DNA restriction fragment containing the viral genome control regions. (b) At low salt concentrations, both subclasses bound to specific and to nonspecific DNA sequences; competition experiments in the presence of nonspecific DNA showed, however, that the affinity of both tumor antigen forms for the viral genome control region was at least 10‐fold higher than their affinity for nonspecific DNA sequences. (c) The binding of the 5–6‐S subclass to viral control region DNA was optimal at 60–80 mM NaCl while specific DNA binding of the 14–16‐S form was optimal at 150–200 mM NaCl; however, binding of the 14–16‐S form to nonspecific DNA sequences was also more resistant to high salt concentrations than that of the 5–6 S form. (d) Both tumor antigen forms bound well to specific and to nonspecific DNA at pH6–6.5; with increasing pH values, binding to nonspecific DNA decreased while binding to specific DNA reached an optimum at pH 7–7.5. Binding of the 14–16‐S form to viral origin DNA was more resistant to pH values above 7.5 than binding of the 5–6‐S form.