Activities of SV40 T antigen necessary for the induction of tetraploid DNA content in permissive CV-1 cells.
To determine the role of SV40 T antigen in stimulating multiple rounds of DNA synthesis in permissive cells, CV-1 cells were transfected with plasmids expressing mutant or wt T antigen in the presence or absence of the SV40 origin of replication. Induction of cells with > G2 DNA content (tetraploid DNA content) and levels of T antigen protein were detected and analyzed by flow cytometry. The mutant T antigen proteins demonstrated the expected phenotypes as determined by immunoprecipitation. Elevated levels of T antigen protein were detected in each transfection, but full-length T antigen alone was responsible for the tetraploid DNA content. The studies show that full-length T antigen with point mutations to reduce binding to the cellular proteins p53 and/or Rb were capable of inducing > G2 DNA content though the induction by these mutants was greatly enhanced by the presence of the SV40 origin of replication. Truncated T antigen (aa 1-259) could induce cells with tetraploid DNA content only in the presence of the SV40 origin of replication and the absence of Rb binding. These studies suggest that multiple functions of T antigen are involved in the stimulation of the second round of cellular DNA synthesis.
Perry
M B
MB
Department of Microbiology, Immunology and Molecular Genetics, Albany Medical College, New York 12208, USA.
Lehman
J M
JM
D000818:Animals; D000952:Antigens, Polyomavirus Transforming / Q000235:genetics* / Q000502:physiology*; D001483:Base Sequence; D002460:Cell Line; D002522:Chlorocebus aethiops; D004247:DNA / Q000235:genetics* / Q000378:metabolism*; D017931:DNA Primers / Q000235:genetics; D005434:Flow Cytometry; D009154:Mutation; D010957:Plasmids / Q000235:genetics; D011123:Polyploidy; D011485:Protein Binding; D018741:Replication Origin; D016160:Retinoblastoma Protein / Q000378:metabolism; D013539:Simian virus 40 / Q000235:genetics / Q000276:immunology / Q000502:physiology; D014162:Transfection; D016159:Tumor Suppressor Protein p53 / Q000378:metabolism; D014779:Virus Replication
D000952:Antigens, Polyomavirus Transforming; D017931:DNA Primers; D016160:Retinoblastoma Protein; D016159:Tumor Suppressor Protein p53; D004247:DNA